# Trends and Advances in Separation and Detection of SSRIs and SNRIs in Biological Matrices

Authors: Ruchita Das; Y. K. Agrawal
Journal: Chromatography Research International
 (2013)

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## Abstract

Nowadays antidepressant drugs like selective serotonin reuptake inhibitors (SSRIs) and selective norepinephrine reuptake inhibitors (SNRIs) represent the first choice in the treatment of moderate to severe depressive illness, various phobias, and personality disorders. In spite of the therapeutic aspects, they often produce very severe and toxic effects in deliberate and accidental cases of poisoning. These are also considered as date-rape drugs used for drugged victims for raping or robbing. Therefore, in recent years, their analyses in different biological matrices for clinical and toxicological analysis purposes has been a target worthy of interest. Thus, the review focuses on recent advancements of various separation techniques like chromatography and electrophoresis that are concernd with the determination of selective serotonin reuptake inhibitor and selective norepinephrine reuptake inhibitor drugs and their metabolites in various biological matrices. In addition to this, a critical discussion on analytical approaches has also been incorporated, suggesting their applicability and limitations for further implementations. Thus, this paper will definitely help in the selection and development of proper analytical methodologies to achieve satisfactory results, better scientific understanding, and test interpretation.


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## Body

## 1. Introduction

Depression is a common and widespread mental disorder affecting millions of people worldwide [1]. It often leads to severe, chronic, and life-threatening illness, affecting the economic and social behaviour of an individual drastically [2, 3]. Even more than 20% of the adult population of the world suffers from depression which may lead to suicidal behaviour [3, 4]. To control such psychic conditions, antidepressant drugs are administered which usually produce effects by blocking the neuronal uptake of biogenic amines [5, 6]. Antidepressants are characterized as 1st, 2nd, and 3rd or the so-called new generation, depending on when they were developed [1]. The new generation antidepressant drugs are the most widespread class of drug and are in fact becoming the drugs of first choice for the treatment of depression, because they are considered to be more potent than other antidepressant groups [5, 7]. The highly prescribed classes of this category are selective serotonin reuptake inhibitors (SSRIs) (fluoxetine (FLX), citalopram (CIT), paroxetine (PAR), sertraline (SER), and fluvoxamine (FLV)) and selective norepinephrine reuptake inhibitors (SNRIs) (venlafaxine (VLF), milnacipran (MCP), and duloxetine (DLX)) (Figure 1) [8]. However, these drugs are created with improved margin of safety but need monitoring due to their side effects and rapid abusiveness, because they are likely to produce mental alertness that increases physical activities [9–12]. Cases were also observed in which suicidal and accidental deaths occurred involving antidepressant drugs that are frequently abused, creating a topic of interest in forensic chemistry and toxicology [13–15]. In addition to this, they were also considered as date-rape drugs, used for the purpose of drugging unsuspected victims for raping or robbing [16].Figure 1
Structures of SSRIs (FLX, CIT, PAR, SER, and FLV) and SNRIs (VLF, MCP, and DLX) group of drugs.Thus, the aim of this paper is to provide a systematic survey of various chromatographic and electrophoretic techniques used for the analysis of SSRIs, SNRIs, and their corresponding metabolites in various biological matrices. These assays consist of three components as the types of biological samples, their pretreatments or isolation, and the techniques of detection and quantitation. All of them influence the accuracy, precision, selectivity, and sensitivity of the analytical methods used. This will simplify the rapid selection of a suitable method for satisfactory clinical and toxicological analyses. To the best of our knowledge, such a review has not yet appeared.


## 2. Separation Techniques Used for the Detection of SSRIs, SNRIs, and Their Metabolites

The separation of analyte of interest forms two basic approaches, the first one is the sample preparation step and the second is the detection of the compound of interest. A number of extraction techniques have been routinely used for the removal of as many as interfering compounds and preconcentration of the analytes. A variety of methods have been published or validated in order to determine the amount of antidepressants in different biological matrices which can fabricate a broad investigating area in the field of therapeutics as well as forensics. These methods offer good precision and accuracy over the entire analytical range, allowing the development of very rapid and efficient methods. Thus, in the next subsections discussion about various analytical methods and sample pretreatment techniques have been incorporated and tabulated.


### 2.1. Liquid Chromatography (LC)

LC provides a wide range of linearity with many detectors as fluorescence detector (FD) [17], ultraviolet-visible (UV-Vis) detector [18], and MS detector [19], maximumly in combination of C18 column with varying ratios of mobile phases.


#### 2.1.1. Mass Detector

LC coupled with single-stage (LC-MS) or tandem mass spectrometry (TMS or LC-MS-MS) is becoming increasingly important in routine laboratory analysis of antidepressant drugs especially from whole blood, plasma/serum, and urine. The alternative matrices such as hair and oral fluid have also given satisfactory results [20]. Moreover, MS has been the most commonly used detector in the determination of antidepressants, the high specificity and the lower limits of detection obtained being the main advantages with this detection technique. With a mass spectrometer, it is possible to monitor only the mass of the analyte which is extremely beneficial in the structural elucidation studies. A precise, sensitive, and high throughput LC-TMS method was developed for simultaneous determination of SER and its primary metabolite, N-desmethylsertraline (DSER) in human plasma. The analytes were extracted from plasma via liquid-liquid extraction (LLE) in methyl tert-butyl ether. The chromatographic conditions were aimed for getting an adequate response, a sharp peak shape, and a short run time per analysis. It was observed that 1 M (2.5 mL) ammonium trifluoroacetate in 750 mL methanol + 250 mL deionized water as the mobile phase provides faster elution, better efficiency, and a peak shape. The use of Betasil C8 chromatographic column helped in the separation and elution of both compounds in a very short time of 1.40 and 1.47 min for SER and N-DSER, respectively. The maximum on-column loading per sample injection was 0.45 ng for both analytes [21]. This volume was considerably less as compared to other reported procedures (injection volume of 20 μL), but the former method helped in maintaining the efficiency and lifetime of the column which in turn gives consistent and reproducible recoveries of analytes [22].Quantitative estimations of VLF and desmethylvenlafaxine (DVLF) were carried out in biological samples obtained from postmortem cases. VLF and one of its active metabolites, DVLF, were identified by LC-MS using atmospheric pressure ionisation (API) electrospray in positive mode, followed by extraction throughn-butyl chloride. Chromatographic separation was performed on C18 column and the mobile phase consisted of ammonia (0.05 M/methanol/tetrahydrofuran (32.5 : 67.0 : 0.5 vol%)) titrated to pH = 10.0 with 12 M formic acid and achieved retention time of 9.1 and 3.5 min for VLF and DVLF, respectively. Moreover, the postmortem tissue concentrations observed were 0.1–36 and <0.05–3.5 mg/L (peripheral blood), <0.05–22 and <0.05–9.9 mg/kg (liver), and <0.05–10 and <0.05–1.5 mg/L (vitreous), <0.05–55 and <0.05–21 mg/L (urine), respectively. Additionally, it was also reported that VLF alone was not responsible for intoxication in the above described postmortem studies. However, it has shown synergism in presence of other drugs like FLX and moclobemide, which could be liable for fatality [23]. An atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) method was developed for the identification and quantitation of CIT, FLV, and PAR in human plasma. Direct analysis of plasma samples was done after dilution in 0.1% formic acid, followed by direct injection into the HPLC system, avoiding a time-consuming sample preparation step. Proteins and other large biomolecules were removed during an online sample cleanup step. The limit of quantitation (LOQ) obtained was below 20 ng/mL which permitted the direct analysis of plasma samples [24].The method of sample introduction to the ionisation source is very important while using a mass detector, and it often depends on the ionisation method being used. The ionisation method to be used should depend on the type of sample under investigation. Different types of ionisation sources were used for quantification of SSRIs and SNRIs. Electron spray ionisation (ESI) mode, in combination with LC, was used for quantification of FLX in plasma samples [25, 26]. As compared to the above electron spray ionisation (ESI) method, APCI mode has also provided higher mass spectrometric response and less susceptibility to ion suppression [27].Fully automated online solid phase extraction (SPE) coupled to LC-TMS was developed and validated for direct analysis of 14 antidepressants including SSRIs and their metabolites in plasma. The integration of sample extraction and HPLC separation into a single system permitted direct injection of the plasma without prior sample pretreatment. An automated online SPE-LC-MS/MS method permitted direct injection of 50 mL of plasma without prior sample pretreatment, ensuring the analysis of the samples in 20 min. Several deuterated drugs were employed as internal standards. For the detection, a tandem mass spectrometer with a triple quadrupole was employed operating in electrospray in the positive ionization mode. Selectivity of the method was achieved by a combination of the retention time and two precursor-product ion transitions for the nondeuterated compounds. The LOQs were estimated as 10 ng/mL for all compounds [28].Recently, a new kind of sample preparation media has been developed as restricted access media (RAM) and possesses a biocompatible surface and adsorption centres which provide very fine extraction in minimal amount of sampling (nearly 1μL). This could be related to miniaturization of chromatographic separation, especially with the improvement of mass sensitivity. By giving minimal sample pretreatment through RAM, FLX was detected in plasma by direct analysis through LC-MS in total run time of 8 min. Calibration curves are constructed under a linear range of 1–250 ng/mL with LOQ = 1 ng/mL. This demonstrates the applicability of a new capillary RAM-LC-MS/MS approach for the detection of antidepressant drugs with less consumption of time, solvent, and samples [29]. Likewise, the integration of the sample extraction apparatus with LC separation and MS detection into a single system permitted the direct injection of diluted urine or plasma samples after filtration. An automated in-tube solid-phase microextraction (SPME) LC-MS method was developed for the analysis of SSRIs and SNRIs in urine and plasma. A hybrid organic-inorganic silica monolith with cyano-ethyl functional groups was prepared and used as a sorbent for in-tube SPME. The total process time was 30 min, and only 30 μL of urine or plasma was required in one analysis cycle, achieving good extraction efficiencies and recoveries (urine and plasma were from 75.2% to 113.0%) with no matrix interferences. Limit of detection (LOD) was found as 0.06–2.84 ng/mL in urine and 0.07–2.95 ng/mL in plasma. This could minimize repetitive work and also eliminates the analyst’s exposure to hazardous and toxic solvents [30].A new type of polymer column (Shim-pack MAYI-ODS) has been used for the separation of SER, FLV, and PAR in human serum by LC-MS/MS. This column is having a unique online dilution by pass channel which can reduce labour and enhances efficiency. The recoveries of the three drugs in human serum were observed as 29.2%–45.7% at 20 ng/mL and 52.0%–53.7% at 80 ng/mL of injection, and each drug had a detection limit of 1–3 ng/mL. Thus, the method enabled direct injection of crude biological samples without complicated pretreatments and without column switching system [31].CIT and its metabolites desmethylcitalopram (DCIT) and didesmethylcitalopram (DDCIT) were separated from plasma samples through SPE, using two types of cartridges during extraction, C8 and Oasis hydrophilic-lipophilic balance cartridges. C8 cartridge has produced good extraction yields, but some interference was also detected due to plasma matrix, while the results obtained with Oasis hydrophilic-lipophilic balance cartridges were better in terms of extraction yield and selectivity. Thus, the later procedure was chosen for extraction, achieving LOQ = 1.5 ng/mL for CIT and DCIT and 2.0 ng/mL for DDCIT [32]. CIT and DCIT were also quantified from neonatal hair samples which suggest gestational exposure of these drugs. A LC-TMS method has been developed and validated for the assay of CIT in neonatal hair samples which also explores the utility of alternative test matrices in detection [33].On the contrary, a simple chemical treatment to the biological sample can also allow the direct injection of the supernatant to the column. A very sensitive HPLC-TMS with ESI method was developed for the simultaneous determination of FLX and norfluoxetine (NFLX) in human plasma. It was observed that the addition of formic acid to mobile phase could improve sensitivity by promoting the ionization of analytes. Acetonitrile was found to be more favourable for the chromatographic separation of FLX and NFLX from matrix than methanol and able to produce high sensitivity (retention time = 3.8 min), lowest background noise, and most stable MS signals (Figures2(a) and 2(b)) [34].(a) The product ion spectra of [M + H]+ of (A) Fluoxetine and (B) Norfluoxetine. (b) Chromatogram of plasma sample spiked with (A) Fluoxetine (3.0 ng/mL) and (B) Norfluoxetine (2.6 ng/mL) (reproduced with the permission from [34]).
(a)
(b)Except for all these proposed methods, a very noble HPLC-TMS method (with single sample/triple injection approach) was developed and validated for the simultaneous determination of forty-eight antidepressants (including SSRIs and SNRIs), antipsychotics, and their pharmacologically active metabolites in serum. The chromatographic separation was performed on a monolithic C18 column (50 mm × 4.6 mm) with methanol gradient and 5 mM acetate buffer (pH 3.9). The sample volume needed for analysis was very less (0.1 mL) which requires only protein precipitation and stepwise dilution for sample preparation. After ESI, positive ion fragments were detected in the multiple reaction monitoring mode. The LOQ was found as 1.0, 2.17, 1.17, 1.07, 0.70 ng/mL for CIT, FLX, FLV, PAR, and SER, respectively. The advantage of the method was to minimize the expenditure for routine determination of large set of individuals with varying drug combinations [35].Newer techniques like ultra-high performance liquid chromatography (UHPLC) have managed to reduce the time analysis and the effectiveness of the separations. An UHPLC-time-of-flight (UHPLC-TOF-MS) method has been proposed for the simultaneous screening and quantification of 52 drugs, including SSRIs in 15 autopsy hair samples, with a total chromatographic run time of 17 min. Hair samples were liquid-liquid extracted with methanol, acetonitrile, ammonium formate (2 mM, 8% acetonitrile, pH and 5.3) overnight at 37°C. The LODs for CIT, FLX, and VLF were found to be 0.01 ng/mL, and for SER and PAR they were found to be 0.02 and 0.05 ng/mL, respectively [36]. Likewise, HPLC-HRMS (high resolution mass spectroscopy) method for simultaneous screening and quantification of 28 drugs, including VLF and FLX, was developed and validated on hair samples. The samples (2.5 mg) were extracted with 200 μL of a mixture of water: acetonitrile: 1 M trifluoroacetic acid (80 : 10 : 10, v/v). Total chromatographic run time was 26 min (VLF = 16.3 min and FLX = 20.5 min), reaching lower limit of quantitation (LLOQs) as low as 0.1–0.5 ng/mg, which are particularly suitable for the forensic determination of selected antidepressants [37].


#### 2.1.2. UV Detector

Owing to the wide applicability of the UV detector, it has been deemed suitable for the development of multiresidue liquid chromatography methods [38]. In order to achieve good chromatographic separation and enhancement of signals, different types of extraction procedures were adopted for the removal of interfering particles such as LLE, SPE, stir bar sorptive extraction (SBSE), and liquid-phase microextraction (LPME) which has given a detection limit up to nano or pico level [39]. Sometimes for multicomponent analysis [40] dual UV wavelength detection [41] has also been used for the identification of compounds. An isocratic reverse phase HPLC (RP-HPLC) method with UV detection was optimised for the determination of SER, FLV, FLX, PAR, CIT, MCP, VLF, and o-desmethylvenlafaxine (ODV) in human serum. The SPE, yielded reproducible recoveries from 75 to 99%, allowed efficient and rapid analysis in minimum amount of time [42].Recently, a newer kind of hollow fibre based liquid-phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of SER from urine and plasma samples prior to its determination by HPLC-UV. This is a kind of miniaturised isolation technique based on the use of disposable, porous, hollow fibres made of polypropylene, which can combine extraction, concentration, and sample cleanup in one step only. The calibration curves were obtained in the range of 5–500μg/L with reasonable linearity (R2 > 0.998). Hence, the method indicates the applicability of hollow fibre microextraction as an excellent cleanup procedure and can be served as a simple and sensitive method for monitoring antidepressant drugs in the biological samples [18].Consequently, there has been an increased effort to develop standardized methods for quantifying psychotropic medications in breast milk which also shows the in utero exposure of these drugs. A method was developed on HPLC-UV for measuring antidepressants in human breast milk. The extraction of drugs was achieved by both techniques as LLE and SPE. Three kinds of assays were performed during analysis, in which assay 1 measured FLX, NFLX, and FLV; assay 2 measured SER and DSER; and assay 3 measured the tricyclic antidepressants. This method was found as highly accurate and precise in measuring the selected antidepressants in human breast milk as it was free from matrix effects which were often encountered in breast milk drug analysis [43].In HPLC analysis, thermal desorption at high temperature creates practical problems such as degradation of the polymer, and furthermore, many nonvolatile compounds cannot be completely desorbed from a fiber. Solvent desorption is thus proposed as an alternative method for SPME-HPLC coupling. An organic solvent (static desorption) or the mobile phase (dynamic desorption) is used to desorb the analytes from the SPME fiber. A high throughput SPME-HPLC-UV method has been developed with high sensitivity, precision, and accuracy and enabled the quantification of CIT, SER, PAR, DLX, and FLX in human plasma, following oral administration. For the extraction of 250 mL of plasma, 4 mL of phosphate buffer (pH 7) was added, and the extraction was carried out at 25°C for 40 min, using a modified fibre obtained by the electrochemical deposition (cyclic voltammetry) of a polypyrrole (PPY) film on a stainless steel wire. The drug liquid desorption was made on mobile phase at 25°C for 15 min. The LOD was found 20 ng/mL for CIT and PAR and 25 ng/mL for FLX and SER, while 16 ng/mL for DLX [44].At times,  a fully automated procedure was also used and allowed online extraction of the drug by HPLC. CIT and its S-enantiomer escitalopram (EsCIT) were quantitatively determined from serum by column-switching HPLC in combination with UV and obtained LOD = 6 ng/mL. The method was found to be sensitive and time and solvent saving [45]. A highly selective HPLC method has been developed to detect secondary amine-bearing antidepressants (PAR and FLX) and their metabolites by using 7,7,8,8 tetracyanoquinodimethane (TCNQ) as a derivatizing agent in plasma samples. The method was based on purple chromogens formed by the displacement reaction of the drugs with a derivatizing agent. The resulting chromogens were directly separated by RP-HPLC. For chromatographic separation, C18 column was used in combination with acetonitrile-water (60 : 40) as the mobile phase and achieved complete separation in 40 min. The LOD observation varied mostly between 240 and 480 ng/mL [46].


#### 2.1.3. Fluorescence Detector

It has also been observed that the fluorescence detection has some advantages over ultraviolet detection. In addition to higher sensitivity, FD is more specific and selective in reducing some interfering peaks. It was the first detection method used for the analysis of CIT and its DCIT. This method required the LLE extraction of the analytes with hexane from alkaline plasma and their derivatization with 9,10-dimethoxyanthracene-2-sulfonate [47]. Similar detection technique has been applied for the detection of CIT and its metabolites in plasma samples with a successive column-switching technique. Plasma samples were injected directly onto a guard column where the analytes were retained and later eluted, employing a six-port valve by the backflush method. The compounds were analyzed at an excitation wavelength of 249 and an emission wavelength of 302 nm [48].SBSE, coupled with HPLC-FD, was used for the quantification of FLX, CIT, and VLF and their active metabolites NFLX, DCIT, DDCIT, and O-DVLF in plasma, urine, and brain tissue samples. For each matrix, the analytical method has been assessed by studying the linearity and the intra- and interday accuracy (89–113%) and precision (RSD < 13%). In addition to this, the improvement in quantification limits was observed which made it as an applicable procedure for all the matrices useful during the forensic analysis. The use of SBSE favours improvement in the LOQ, as the surface of extraction is higher than microextraction fibers (Figure3) [49].Figure 3
Chromatograms obtained from plasma and brain tissue of rat and urine of human.The retention time of the compounds was Fluoxetine (FLX) = 21.2 min and its metabolite norfluoxetine (NFLX) = 16.1 min, Citalopram (CIT) = 10.6 min and its metabolites o-desmethylvenlafaxine (ODV) = 3.1 min, didesmethylcitalopram (DDCIT) = 7.0 min, venlafaxine (VLF) = 7.8 min, and desmethylcitalopram (DCIT) = 8.6 min. (Reproduced with the permission from [49])Likewise, a quantitative analysis of PAR and MCP has been achieved using an isocratic RP-HPLC combined with FD from human serum samples which reported good linearity and correlation coefficients (r=0.999). Extraction was done with LLE which gave high efficiency, selectivity, and simplicity for the given method and observed less elution time for PAR (7.2 min) [50]. This is very low as compared to the above reported procedure (15.3 min) in spite of the use of high throughput SPE [42].Similarly, FLX was detected in serum using 4-chloro-7-nitro-2,1,3-benzofurazan as a precolumn derivatizing agent in order to improve the sensitivity of the assay. The chromatographic separation was performed on C18 column, and the fluorescence derivatives of the drugs were monitored at the excitation and emission wavelengths of 470 and 537 nm, respectively. Achieved LOQ (0.5 ng/mL) within 6 min, which was more satisfactory than other reported methods, which allowed the determination of low blood FLX levels also. It has been observed that the formations of derivative products were highly dependent on the pH of buffer solution, time, temperature, concentration of the labelling agent, and polarity of the medium. [51]. Other derivatizing agents have also been used to improve the sensitivity of the assay as 4-fluoro-7-nitrobenzofurazan [52], fluorescamine [53], and dansyl chloride [54]. However, while using fluorescamine and dansyl derivatives, there might be a possibility of poor photo stability and band broadening of the resulted peaks. Table 1 represents the LC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.Table 1
Liquid chromatographic methods developed for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction method
Detector system
Sensitivity
References
Mass detector
Plasma
SER andN-DSER
LLE
TMS
LOQ = 0.5 ng/mL
[21]
Plasma
FLX and NFLX
LLE
ESI, SIM*
LOQ = 2.5μg/mL for FLX and 10 μg/mL NFLX
[25]
Plasma
FLX
SBSE
ESI, SIM
LOQ = 30 pg/mL (liquid desorption) and 1.37 pg/mL (thermal desorption)
[26]
Hair
CIT, DCIT, and DDCIT
LLE
TMS
LOD = 10 pg/mgLOQ = 20 pg/mg
[33]
Plasma
VLF,O-DVLF, N-DVLFN,O-didesmethylvenlafaxine (DDVLF)
LLE
MS
LOD = 0.4, 0.2, 0.3, and 0.2 ng/mL andLLOQ = 3.5, 2.2, 2.7, and 1.9 ng/mL for VLF, O-DVLF, N-DVLF, and DDVLF, respectively
[75]
Plasma
SER
SPE
Triple stage(ESI, SRM)
LOQ = 0.5 ng/mL
[76]
Oral fluid and plasma
FLX, NFLX PAR, SER, FLV, CIT, and VLF
Automated SPE
TMS (ESI), with triple quadrupole
LOQ = 2 ng/mL in oral fluid, 2 ng/mL in plasma (except FLV 10 ng/mL and NFLX 4 ng/mL)
[77]
Plasma
FLX and NFLX
Automated SPE
Triple stage(ESI, SRM)
LLOQ = 0.5 ng/mL
[78]
Plasma
FLX and NFLX
Online extraction using column switching
ESI, SIM
LOQ = 25 ng/mL
[79]
Plasma
VLF andO-DVLF
SPE
TMS, SRM
LOQ = 3 ng/mL for VLF, 6 ng/mL for DVLF
[80]
Plasma
CIT
LLE
ESI, SIM
LOQ = 0.50 ng/mL
[81]
Plasma
CIT and DCIT
Deproteinization
MS (SIM)
LOQ = 0.25 to 0.5 ng/mL
[82]
Plasma
FLV, PAR, and MCP
SPE
QMS-(SSI)*
LOD = 0.05 to 0.13μg/mL and LOQ = 0.10 to 0.20 μg/mL
[83]
Plasma
FLX and NFLX
LLE
TMS (ESI)
LOQ = 0.15 ng/mL
[84]
Plasma
FLX, CIT, PAR, and VLF
SPE
MS (ESI)
LOD, FLX = 0.5, CIT = 0.3, PAR = 0.3, and VEN = 0.1 ng/mL
[85]
Blood
FLX, PAR, SER, FLV, CIT, VLF, NFLX, DCIT, DDCIT, and DVLF
LLE
TMS
LOD = 5–500 ng/mL and LOQ = 5 ng/mLLOD = 20–2000 ng/mL for VLF and DVLF LOQ = 20 ng/mL for VLF and DVLF
[86]
UV detector
Urine and plasma
SER
HF-LPME
UV
LOD = 0.5μg/L LOQ = 2.3 μg/L
[18]
Plasma
CIT and DCIT
LLE
UV
LOD = 5 ng/mL
[38]
Plasma
CIT, SER, FLX, PAR, and DLX
SBSE
UV
LOD = 20 ng/mL for CIT and PAR, 25 ng/mL for FLX and SER and 16 ng/mL for DLX
[44]
Plasma
FLX, SER, PAR, and CIT
LLE
UV
LOQ = 10 ng/mL for FLX, SER, and PAR and 5 ng/mL for CIT
[87]
Plasma and urine
FLX, PAR, and VLF
SPE
UV
LOD = 0.2–0.6 ng/μL in plasma and 0.1–0.8 ng/μL in urine
[88]
Plasma
CIT, FLX, PAR
SPE
UV
LOQ = 0.05 to 0.26 mM
[89]
Plasma
CIT, DCIT, DDCIT, FLV, PAR, SER,FLX, NFLX, VLF, andO-DVLF
LLE
UV
LOQ = 25 ng/mL,   100 ng/mL for VLF andO-DVLF
[90]
Plasma
R- and S-CIT, R- and S-DCIT, and R- and S-DDCIT
LLE
UV
LOD > 2 ng/mL
[91]
Fluorescence detector
Plasma
FLX and NFLX
LLE
FD
LLOD = 3 mg/L
[17]
Plasma
SER and DSER
SPE
FD
LOQ = 1.0 ng/mL  and LOD = 0.3 ng/mL
[22]
Plasma
CIT, DCIT, DDCIT
SPE
FD
LOQ = 1.5 to 2 ng/mL
[32]
Plasma, urine, and brain tissue
CIT, DCIT, DDCIT, FLX, NFLX, VLF, andO-DVLF
SBSE
FD
LOQ = 0.2 to 2 mg/L in plasma, 2 to 20 ng/g in brain tissue, and 1 to 10 mg/L in urine
[49]
Serum
SER
LLE
FD
LOQ up to 2 ng/mL
[53]
Plasma
CIT, DCIT, DDCIT, PAR, and PAR metabolites
SPE
FD
LOQ = 0.025 to 0.12 mmol/L
[92]
Serum
CIT, FLX, and NFLX
LLE
FD
LOQ = 50 mg/L
[93]
Plasma
FLX and NFLX
HF-LPME
FD
LOQ = 5 ng/mL
[89]
Plasma
CIT, DCIT, and DDCIT
SPE
FD
LOD = 0.8 nmol/L
[94]
Diode array detector
Plasma
FLX, NFLX, SER, PAR, CIT, FLV, VLF, MCP, and DVLF
LLE
DAD
LOD = 2.5 to 5 ng/mL
[95]
Urine
CIT, DCIT, DDCIT, FLX, and NFLX
SPME
DAD
LOD = 0.01–0.09 mg/L
[96]
Abbreviations*: SIM: Single Ion Monitoring; QMS: Quadrupole Mass Spectrometry; SSI: Sonic Spray Ionisation.


### 2.2. Gas Chromatography (GC)

The use of GC with varying detectors has proved to be a powerful tool in the area of underivatized and derivatized drug analysis from biological extracts. The following are the different detectors used in combination with gas chromatography for the analysis of SSRIs and SNRIs in a biological matrix.


#### 2.2.1. Mass Detector (MS)

A method was developed based on a capillary gas chromatography with mass spectrometry detection in selected ion monitoring (SIM) mode for the analysis of CIT, FLX, DCIT, DDCIT, and NFLX in urine samples. An optimized SPE procedure was applied which provided a preconcentration factor of 10, and no derivatization step was required [55].In order to achieve a high degree of separation for SER, PAR, CIT, DCIT, DDCIT, and DSER in a plasma sample, reextraction was done at varying pH, resulting in less interference from endogenous compounds. Such kinds of GC separations were performed using a cross-linked 30 m DB-35 ms silica capillary column, and helium (He) was used as a carrier gas (flow rate = 1.5 mL/min), with a mass spectroscopic measurement in ESI mode. The simultaneous chromatographic separation was achieved after the derivatization of analytes with reagent N-methyl-bis(trifluoroacetamide) [56]. SPME-GC has been used for the determination of SSRIs in urine samples. Fibers of 65 mm polydimethylsiloxane-divinylbenzene (PDMS-DVB) were used for the determination of the target SSRIs after simple in situ derivatization by acetylation of FLV, FLX, and SER. A 1.5 g aliquot of sodium chloride was then added to the samples. The vial was sealed with an aluminum cap and a teflon faced septum. It was then immersed in a water bath at 100°C and left to equilibrate for 5 min. To perform the extraction, the SPME fiber was exposed to the magnetically stirred sample for 30 min. The fiber was then immediately inserted into GC injection port and analysis was performed. Desorption time was set at 3 min. The time required for SPME step and for GC analysis (30 min each) enabled high throughputness [57].Derivatization is an important process which helps in large-scale screening of varieties of drug. This helps in the conversion of thermally labile and nonvolatile analytes to a suitable form for GC analysis, which has extended its use in the detection of antidepressant drugs and their metabolites. The separation of VLF, FLV, FLX, and CIT in blood samples was obtained by GC-MS operating in select ion monitoring (SIM) mode. Analytes were isolated by high yield LLE followed by derivatization throughtert-butyldimethylsilylation (MTBSTFA) and tri-methylsilylation (MSTFA) reagents. Thus, the analytes were analysed in tert-butyldimethylsilylated (TBDMS) and tri-methylsilylated forms (TMS) by GC-MS. Due to superior derivatization power and intensive derivatives, silylation was believed to have the best properties for large-scale screening with a variety of substances having structural differences. The method is feasible for highly sensitive, reliable, and routinely performed forensic toxicological analysis that produced satisfactory results with different concentration ranges of analytes in small sample volume [58]. In general, the derivatizing agents mostly preferred during GC separation of antidepressant drugs were N-methyl-N-trimethylsilyl-trifluoroacetamide for silylation, acetic anhydride-pyridine for acetylation, and trimethylammonium hydroxide for methylation [59].The identification and quantification of psychoactive drugs like antidepressants can be obtained using alternative biological matrices like oral fluid. Analyses of SER and PAR were obtained with GC-MS(-MS), operating in SIM mode after their SPE from oral fluid. Due to the basic nature of these drugs, eluting solvent used in SPE is a mixture of dichloromethane: isopropanol: ammonium (80 : 20 : 2, v/v/v). The chromatographic separation was performed on methylsilicone capillary column, and analytes were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and obtained retention time of 12.16 (SER) and 12.49 min (PAR). The method showed the applicability of alternative test matrices like oral fluid which have been extensively and successfully used to access acute and chronic exposure of drugs of abuse [60]. Similarly, in a suicidal case of multiple drug ingestion, CIT was identified and quantified in the postmortem samples by GC-MS. The concentrations of CIT found in femoral blood, heart blood, and urine were 0.88 mg/L, 1.16 mg/L, and 0.9 mg/L, respectively. This method also shows its applicability in the determination of CIT in postmortem tissue samples like liver, brain, gastric content, and so forth [61].Moreover, full-scan EI ionization mode is also considered as the reference technique for drug screening procedures since it plays an important role in the analysis of drugs in forensic toxicology and doping control. The main advantage of this technique is that it provides comparable GC-MS spectra on all commercially available instruments since the standardized ionization EI mode produces unique and uniform mass fragmentation patterns. As a consequence, large mass spectral libraries have been constructed which enable the identification of unknown compounds [62]. Table 2 represents the GC methods that appeared in the literature for the determination of new generation antidepressant drugs and their metabolites.Table 2
Gas chromatographic methods developed on for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction       method
Detector system
Sensitivity
References
Mass detector
Urine
FLX, VLF, SER, and CIT
SPE
EI-MS
LOD, 2, 0.02, 1.25,  and 10μg/mL for FLX, VLF, SER, and CIT
[16]
Plasma
CIT, DCIT, DDCIT, FLX, and NFLX
SPE
MS
LOD = 0.7  to 33.6μg/mL
[55]
Plasma
CIT, DCIT, DDCIT, SER, DSER, and PAR
LLE
MS
LOQ = 2 ng/mL for CIT and PAR, 1 ng/mL for SER, 0.5 ng/mL for DSER, DCIT, and DDCIT
[56]
Urine
FLV, FLX, SER, VLF, and CIT
SPME
MS (ESI mode)
LOD < 0.4 ng/mL
[57]
Blood
VLF, FLX, FLV, CIT, and SER
LLE
MS (ESI in SIM mode)
LOD = <10, 25, 25, <10, 4 ng/mL, and LOQ = 25, 50, 25, 25, 50, 25, 20 ng/mL for VLF, FLV, LX, CIT, and SER, respectively
[58]
Oral fluid
SER
SPE
MS in SIM mode.
LOD = 18.6 ng/mL
[60]
Plasma
VLF, CIT, FLX, FLV, SER, PAR, DFLX, DSER, DCIT, and DDCIT
SPE
Mass (ESI and Chemical Ionisation  (PCI*/NCI* source) in SIM mode)
LOQ = 5 to 12.5 ng/mL in ESI and PICI mode and LOQ = 1 to 6.25 ng/mL in NCI mode
[97]
Plasma
CIT, DCIT, and DDCIT
LLE
MS
LOQ = 1 to 2 ng/mL
[98]
Other detectors
Serum
CIT and DCIT
LLE
NPD
LOQ = 10–20 mg/L
[63]
Blood
CIT
SPE
NPD
LOQ = 70 ng/mL
[99]
Urine
CIT, FLX, FLV, SER, and PAR
SPE
FID
LOQ = 91.7,  64.9, 100.3, and 61.4 and 95.1μg/L for CIT, FLX, FLV, SER, and PAR, respectively
[100]
Plasma
CIT, PAR, DLX, FLX, and SER
In-tube SPME
UV
LOQ = 50 to 500 ng/mL
[101]
Abbreviations*: PCI: Positive Chemical Ionization, NCI: Negative Chemical Ionization.


#### 2.2.2. Nitrogen Phosphorous Detector (NPD)

Despite the differences in chemical structures of FLX, SER, CIT, VLF, DCIT, DSER, and NFLX,  were successfully assayed in serum samples after LLE, followed by GC-NPD. It has been observed that for GC procedure, polarity of extracting solvent (proportion of isoamyl alcohol in the mixture with hexane) was one of the most important variables influencing extractions. An added factor, which was also considered during analysis, is the nitrogen containing structures of analytes and responsible for the high sensitivity obtained using nitrogen phosphorous detector (NPD) (helps in minimisation of interferences). The method was reported with good linearity (20 to 400 mg/L) and correlation coefficients (r=0.998). The specificity of the method can help during the toxicological screening of antidepressant drugs [63]. Even stereoselective determination of FLX and NFLX enantiomers were obtained by using the two-dimensional capillary gas liquid chromatography with NPD that allows the separation of racemic mixtures of the drugs in plasma or serum. A three-step LLE was used for sample preparation and provided linear calibration between 5–250 ng/mL for (R)- and (S)-FLX and 15–250 ng/mL for (R)- and (S)-NFLX, with LOD = 1.5 ng/mL and 6 ng/mL, respectively [64].A comparative study was done for two types of extraction procedures regarding their recovery, precision, sensitivity, matrix purification efficiency, and less solvent consumption for the detection of SER and VLF in blood samples using GC-NPD. First procedure consists of the employment of chem elut column (diatomaceous earth) based on the principle of solid-liquid absorption extraction and closely related to conventional LLE, while in the second bond, elut certify column was used with mixed SPE having reversed-phase and cation-exchange sorbents. Recoveries of the compounds using chem elut columns at 500 ng/mL were in the range of 28–74% and with LOD and LOQ ranging from 39 to 153 ng/mL and from 128 to 504 ng/mL, respectively, while recoveries of the compounds using bond elut certify columns at 500 ng/mL were in the range of 64–86% and with LODs and LOQs ranging from 21 to 100 ng/mL and from 70 to 330 ng/mL, respectively. Thus, the results suggested that the use of reversed-phase and cation-exchange sorbent bond elut certify columns is more satisfactory for the screening of the selected drugs [65]. In a similar kind of study, the use of mixed-mode bonded-silica bond elut certify columns had shown advantages rather than chem elut columns, in terms of recoveries, cleaner extracts, sensitivity, precision and solvent consumption. It has also been explained that the chem elut column has diatomaceous earth support which was a hydrophilic packing porous material acted as a support for the aqueous phase. Its high surface area ensured that the organic eluents remain immiscible with the aqueous matrix, eliminating emulsion problems and facilitating an efficient interaction between sample and organic solvents, while bond elut certify column was a mixed-mode bonded silica extraction support containing hydrophobic chains and strong cation-exchange moieties. This gave the ability to retain acidic, neutral, basic, and amphoteric drugs on one column which is quite advantageous in the field of forensic toxicology [66]. Table 2 represents the GC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.


### 2.3.  Electrodriven Methods

These are efficient and reliable analytical methods applied for the determination of enantiomers. Enantioseparations by capillary electrophoresis (CE) are generally achieved by adding a chiral selector to the running buffer, which reduces the cost of analysis with respect to some techniques such as HPLC chiral column. Cyclodextrins (CDs) are among the most prevalent selectors used in chiral CE. CE is one of the advantageous techniques used for the determination of small molecules in biological fluids which provides higher separation efficiency and selectivity in low sampling and solvent consumption. However, when biological fluids are injected directly into the capillary of CE unit, problems such as clogging of the capillary by particulate matter, irreversible binding of proteins, and peak distortion can occur because of the high salt concentration. Therefore, proper sample preparation or extraction is a must before analysis. The separation of SER, FLX, and FLV was described by CE with fully integrated SPE procedure. The SPE module was placed directly at the inlet of the capillary, and a mixture of all the analytes is extracted from aqueous solution by applying a simple pressure rinse. Under pressure-driven conditions, efficient elution was observed by using 50 mM phosphate buffer (pH = 3.5) in acetonitrile (20/80, v/v). It was observed that neutral monolith material in SPE gave an unstable electroosmotic flow, resulting in poor separation and elution. On the other hand, the addition of ionisable monomer (3-sulfopropyl methacrylate) to the SPE module has given improved and stable electroosmotic flow. This demonstrates the potential of inline SPE-CE for sensitive analysis of these drugs [67].CE, combined with LPME (porous polypropylene hollow fibres), was evaluated for chiral determination of CIT in plasma. The CIT enantiomers (R- and S-CIT) were extracted from 0.5 mL of plasma in alkaline medium (0.25 mL of 2 M NaOH). The unionised analytes were extracted into di-n-hexyl ether, impregnated in the pores of the hollow fibre, and into an acidic solution inside the hollow fibre and obtained excellent recoveries for both enantiomers (80%). This resulted in a three-phase system where the extracts were aqueous and hence directly compatible with the CE system. LOQ and LOD were found as 11.2 ng/mL and 3 ng/mL for both enantiomers, respectively. It has also been observed that the technique LPME-CE was a promising combination for the analysis of racemic drugs present in low concentrations in biological matrices [68].Similar kind of extraction procedure was also adopted in combination with CE, for the determination of CIT and its main metabolite N-DCIT in human plasma. The samples were made strongly alkaline which support proper extraction of the basic drugs like CIT. The drug and its metabolite were extracted into hexyl ether, impregnated in the pores of polypropylene hollow fibre, and into 25μL of 20 mM phosphate buffer (pH 2.75) present inside the hollow fibre (acceptor phase). This acceptor phase reduces the possibility of the majority of endogenic substances to penetrate inside hexyl ether layer. The instrument was operated at 20 kV, generating a current level of approximately 85 mA. LOQ and LOD for citalopram and N-desmethylcitalopram were found to be 16.5 ng/mL and 18 ng/mL and 5 ng/mL and 5.5 ng/mL, respectively [69].Aqueous and nonaqueous CE-MS in ESI mode were investigated for the separation of VLF and its metabolites. The separation of analytes at basic pH was found effective in resolving the drugs, but created considerable peak tailing. To overcome electrostatic interactions between analytes and silanol groups, experiments were carried out at acidic pH which lead to fine separation. Resolution was achieved in less than 3.5 min by the use of fused-silica capillary and a methanol-acetonitrile mixture (20/80 v/v) containing 25 mM ammonium formate and 1 M formic acid. It was also observed that the selectivity, efficiency, and separation time were greatly affected by the organic solvent composition. Nonaqueous CE was found as an appropriate alternative to resolve VLF and its metabolites simultaneously, as the electric current generated in nonaqueous medium was very low and allowed baseline resolution of VLF and its metabolites in 0.7 min only. However, the electric field was further increased by reducing the capillary length [70].Micellar electrokinetic chromatography (MEKC) with DAD was employed for the determination of CIT, SER, PAR, VLF, MCP, FLX, and their metabolites in urine samples. Here, the separation occurs on the basis of hydrophobic interaction with surfactant micelles. Separation and determination were optimised on an uncoated fused-silica capillary. The migration buffer consisted of 20 mM sodium borate at pH 8.5 with 20 mM sodium dodecyl sulphate and 15% isopropanol, at an operating voltage of 25 kV and column temperature at 40°C. Injection in the capillary was performed in hydrodynamic mode (0.5 psi, 15 s) and obtained migration time less than 11 min. Samples (1 mL) were extracted with diethyl ether (5 mL) at pH 9.6 and reconstituted in diluted migration buffer. LOD and LOQ were in ranges between 10 and 20 ng/mL and between 20 and 30 ng/mL, respectively, for all the molecules. This method allowed the determination of some of these analytes in biological fluids (blood and urine) obtained from postmortem cases that suggest the applicability of MEKC in forensic toxicological testing [71].Recently, citalopram and its main active metabolite were analysed in human urine samples through a new MEKC method. The samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C18 cartridge and by eluting the compounds with methanol. Best results were obtained by using 15 mM borate buffer (pH 9.2), containing 20 mM sodium dodecyl sulphate and 12% (v/v) 2-propanol as the background electrolyte. The separation was performed through a fused silica capillary with the application of hydrodynamic injection (pressure = 3.45 kPa) and 30 kV of separation voltage. Under these conditions, the migration times were observed in the range of 3.0 to 8.0 min, and the LODs were observed between 12.5 and 25 ng/mL, respectively [72].Since these compounds were produced in ionic form they can also be separated from capillary isotachophoresis (CITP). This is a technique used to separate charged particles, by using a discontinuous electrical field to create sharp boundaries between the sample constituents, thus creating separation. Conductivity detection of SER, CIT, FLX, and FLV has been done using CITP technique. This shows that it is a simple and quick method for the determination of these drugs [73]. An online preconcentration method, cation-selective exhaustive injection, and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC), with DAD, have been developed for the analysis of SSRIs. Under the optimized separation parameters, the enrichment factors for these five compounds when using CSEI-sweeping-MEKC fell within the range from 5.7 × 104 to 1.2 × 105, achieved LODs for the five SSRIs in human plasma between from 0.35 to 1.5 ng/mL [74].


## 2.1. Liquid Chromatography (LC)

LC provides a wide range of linearity with many detectors as fluorescence detector (FD) [17], ultraviolet-visible (UV-Vis) detector [18], and MS detector [19], maximumly in combination of C18 column with varying ratios of mobile phases.


### 2.1.1. Mass Detector

LC coupled with single-stage (LC-MS) or tandem mass spectrometry (TMS or LC-MS-MS) is becoming increasingly important in routine laboratory analysis of antidepressant drugs especially from whole blood, plasma/serum, and urine. The alternative matrices such as hair and oral fluid have also given satisfactory results [20]. Moreover, MS has been the most commonly used detector in the determination of antidepressants, the high specificity and the lower limits of detection obtained being the main advantages with this detection technique. With a mass spectrometer, it is possible to monitor only the mass of the analyte which is extremely beneficial in the structural elucidation studies. A precise, sensitive, and high throughput LC-TMS method was developed for simultaneous determination of SER and its primary metabolite, N-desmethylsertraline (DSER) in human plasma. The analytes were extracted from plasma via liquid-liquid extraction (LLE) in methyl tert-butyl ether. The chromatographic conditions were aimed for getting an adequate response, a sharp peak shape, and a short run time per analysis. It was observed that 1 M (2.5 mL) ammonium trifluoroacetate in 750 mL methanol + 250 mL deionized water as the mobile phase provides faster elution, better efficiency, and a peak shape. The use of Betasil C8 chromatographic column helped in the separation and elution of both compounds in a very short time of 1.40 and 1.47 min for SER and N-DSER, respectively. The maximum on-column loading per sample injection was 0.45 ng for both analytes [21]. This volume was considerably less as compared to other reported procedures (injection volume of 20 μL), but the former method helped in maintaining the efficiency and lifetime of the column which in turn gives consistent and reproducible recoveries of analytes [22].Quantitative estimations of VLF and desmethylvenlafaxine (DVLF) were carried out in biological samples obtained from postmortem cases. VLF and one of its active metabolites, DVLF, were identified by LC-MS using atmospheric pressure ionisation (API) electrospray in positive mode, followed by extraction throughn-butyl chloride. Chromatographic separation was performed on C18 column and the mobile phase consisted of ammonia (0.05 M/methanol/tetrahydrofuran (32.5 : 67.0 : 0.5 vol%)) titrated to pH = 10.0 with 12 M formic acid and achieved retention time of 9.1 and 3.5 min for VLF and DVLF, respectively. Moreover, the postmortem tissue concentrations observed were 0.1–36 and <0.05–3.5 mg/L (peripheral blood), <0.05–22 and <0.05–9.9 mg/kg (liver), and <0.05–10 and <0.05–1.5 mg/L (vitreous), <0.05–55 and <0.05–21 mg/L (urine), respectively. Additionally, it was also reported that VLF alone was not responsible for intoxication in the above described postmortem studies. However, it has shown synergism in presence of other drugs like FLX and moclobemide, which could be liable for fatality [23]. An atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) method was developed for the identification and quantitation of CIT, FLV, and PAR in human plasma. Direct analysis of plasma samples was done after dilution in 0.1% formic acid, followed by direct injection into the HPLC system, avoiding a time-consuming sample preparation step. Proteins and other large biomolecules were removed during an online sample cleanup step. The limit of quantitation (LOQ) obtained was below 20 ng/mL which permitted the direct analysis of plasma samples [24].The method of sample introduction to the ionisation source is very important while using a mass detector, and it often depends on the ionisation method being used. The ionisation method to be used should depend on the type of sample under investigation. Different types of ionisation sources were used for quantification of SSRIs and SNRIs. Electron spray ionisation (ESI) mode, in combination with LC, was used for quantification of FLX in plasma samples [25, 26]. As compared to the above electron spray ionisation (ESI) method, APCI mode has also provided higher mass spectrometric response and less susceptibility to ion suppression [27].Fully automated online solid phase extraction (SPE) coupled to LC-TMS was developed and validated for direct analysis of 14 antidepressants including SSRIs and their metabolites in plasma. The integration of sample extraction and HPLC separation into a single system permitted direct injection of the plasma without prior sample pretreatment. An automated online SPE-LC-MS/MS method permitted direct injection of 50 mL of plasma without prior sample pretreatment, ensuring the analysis of the samples in 20 min. Several deuterated drugs were employed as internal standards. For the detection, a tandem mass spectrometer with a triple quadrupole was employed operating in electrospray in the positive ionization mode. Selectivity of the method was achieved by a combination of the retention time and two precursor-product ion transitions for the nondeuterated compounds. The LOQs were estimated as 10 ng/mL for all compounds [28].Recently, a new kind of sample preparation media has been developed as restricted access media (RAM) and possesses a biocompatible surface and adsorption centres which provide very fine extraction in minimal amount of sampling (nearly 1μL). This could be related to miniaturization of chromatographic separation, especially with the improvement of mass sensitivity. By giving minimal sample pretreatment through RAM, FLX was detected in plasma by direct analysis through LC-MS in total run time of 8 min. Calibration curves are constructed under a linear range of 1–250 ng/mL with LOQ = 1 ng/mL. This demonstrates the applicability of a new capillary RAM-LC-MS/MS approach for the detection of antidepressant drugs with less consumption of time, solvent, and samples [29]. Likewise, the integration of the sample extraction apparatus with LC separation and MS detection into a single system permitted the direct injection of diluted urine or plasma samples after filtration. An automated in-tube solid-phase microextraction (SPME) LC-MS method was developed for the analysis of SSRIs and SNRIs in urine and plasma. A hybrid organic-inorganic silica monolith with cyano-ethyl functional groups was prepared and used as a sorbent for in-tube SPME. The total process time was 30 min, and only 30 μL of urine or plasma was required in one analysis cycle, achieving good extraction efficiencies and recoveries (urine and plasma were from 75.2% to 113.0%) with no matrix interferences. Limit of detection (LOD) was found as 0.06–2.84 ng/mL in urine and 0.07–2.95 ng/mL in plasma. This could minimize repetitive work and also eliminates the analyst’s exposure to hazardous and toxic solvents [30].A new type of polymer column (Shim-pack MAYI-ODS) has been used for the separation of SER, FLV, and PAR in human serum by LC-MS/MS. This column is having a unique online dilution by pass channel which can reduce labour and enhances efficiency. The recoveries of the three drugs in human serum were observed as 29.2%–45.7% at 20 ng/mL and 52.0%–53.7% at 80 ng/mL of injection, and each drug had a detection limit of 1–3 ng/mL. Thus, the method enabled direct injection of crude biological samples without complicated pretreatments and without column switching system [31].CIT and its metabolites desmethylcitalopram (DCIT) and didesmethylcitalopram (DDCIT) were separated from plasma samples through SPE, using two types of cartridges during extraction, C8 and Oasis hydrophilic-lipophilic balance cartridges. C8 cartridge has produced good extraction yields, but some interference was also detected due to plasma matrix, while the results obtained with Oasis hydrophilic-lipophilic balance cartridges were better in terms of extraction yield and selectivity. Thus, the later procedure was chosen for extraction, achieving LOQ = 1.5 ng/mL for CIT and DCIT and 2.0 ng/mL for DDCIT [32]. CIT and DCIT were also quantified from neonatal hair samples which suggest gestational exposure of these drugs. A LC-TMS method has been developed and validated for the assay of CIT in neonatal hair samples which also explores the utility of alternative test matrices in detection [33].On the contrary, a simple chemical treatment to the biological sample can also allow the direct injection of the supernatant to the column. A very sensitive HPLC-TMS with ESI method was developed for the simultaneous determination of FLX and norfluoxetine (NFLX) in human plasma. It was observed that the addition of formic acid to mobile phase could improve sensitivity by promoting the ionization of analytes. Acetonitrile was found to be more favourable for the chromatographic separation of FLX and NFLX from matrix than methanol and able to produce high sensitivity (retention time = 3.8 min), lowest background noise, and most stable MS signals (Figures2(a) and 2(b)) [34].(a) The product ion spectra of [M + H]+ of (A) Fluoxetine and (B) Norfluoxetine. (b) Chromatogram of plasma sample spiked with (A) Fluoxetine (3.0 ng/mL) and (B) Norfluoxetine (2.6 ng/mL) (reproduced with the permission from [34]).
(a)
(b)Except for all these proposed methods, a very noble HPLC-TMS method (with single sample/triple injection approach) was developed and validated for the simultaneous determination of forty-eight antidepressants (including SSRIs and SNRIs), antipsychotics, and their pharmacologically active metabolites in serum. The chromatographic separation was performed on a monolithic C18 column (50 mm × 4.6 mm) with methanol gradient and 5 mM acetate buffer (pH 3.9). The sample volume needed for analysis was very less (0.1 mL) which requires only protein precipitation and stepwise dilution for sample preparation. After ESI, positive ion fragments were detected in the multiple reaction monitoring mode. The LOQ was found as 1.0, 2.17, 1.17, 1.07, 0.70 ng/mL for CIT, FLX, FLV, PAR, and SER, respectively. The advantage of the method was to minimize the expenditure for routine determination of large set of individuals with varying drug combinations [35].Newer techniques like ultra-high performance liquid chromatography (UHPLC) have managed to reduce the time analysis and the effectiveness of the separations. An UHPLC-time-of-flight (UHPLC-TOF-MS) method has been proposed for the simultaneous screening and quantification of 52 drugs, including SSRIs in 15 autopsy hair samples, with a total chromatographic run time of 17 min. Hair samples were liquid-liquid extracted with methanol, acetonitrile, ammonium formate (2 mM, 8% acetonitrile, pH and 5.3) overnight at 37°C. The LODs for CIT, FLX, and VLF were found to be 0.01 ng/mL, and for SER and PAR they were found to be 0.02 and 0.05 ng/mL, respectively [36]. Likewise, HPLC-HRMS (high resolution mass spectroscopy) method for simultaneous screening and quantification of 28 drugs, including VLF and FLX, was developed and validated on hair samples. The samples (2.5 mg) were extracted with 200 μL of a mixture of water: acetonitrile: 1 M trifluoroacetic acid (80 : 10 : 10, v/v). Total chromatographic run time was 26 min (VLF = 16.3 min and FLX = 20.5 min), reaching lower limit of quantitation (LLOQs) as low as 0.1–0.5 ng/mg, which are particularly suitable for the forensic determination of selected antidepressants [37].


### 2.1.2. UV Detector

Owing to the wide applicability of the UV detector, it has been deemed suitable for the development of multiresidue liquid chromatography methods [38]. In order to achieve good chromatographic separation and enhancement of signals, different types of extraction procedures were adopted for the removal of interfering particles such as LLE, SPE, stir bar sorptive extraction (SBSE), and liquid-phase microextraction (LPME) which has given a detection limit up to nano or pico level [39]. Sometimes for multicomponent analysis [40] dual UV wavelength detection [41] has also been used for the identification of compounds. An isocratic reverse phase HPLC (RP-HPLC) method with UV detection was optimised for the determination of SER, FLV, FLX, PAR, CIT, MCP, VLF, and o-desmethylvenlafaxine (ODV) in human serum. The SPE, yielded reproducible recoveries from 75 to 99%, allowed efficient and rapid analysis in minimum amount of time [42].Recently, a newer kind of hollow fibre based liquid-phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of SER from urine and plasma samples prior to its determination by HPLC-UV. This is a kind of miniaturised isolation technique based on the use of disposable, porous, hollow fibres made of polypropylene, which can combine extraction, concentration, and sample cleanup in one step only. The calibration curves were obtained in the range of 5–500μg/L with reasonable linearity (R2 > 0.998). Hence, the method indicates the applicability of hollow fibre microextraction as an excellent cleanup procedure and can be served as a simple and sensitive method for monitoring antidepressant drugs in the biological samples [18].Consequently, there has been an increased effort to develop standardized methods for quantifying psychotropic medications in breast milk which also shows the in utero exposure of these drugs. A method was developed on HPLC-UV for measuring antidepressants in human breast milk. The extraction of drugs was achieved by both techniques as LLE and SPE. Three kinds of assays were performed during analysis, in which assay 1 measured FLX, NFLX, and FLV; assay 2 measured SER and DSER; and assay 3 measured the tricyclic antidepressants. This method was found as highly accurate and precise in measuring the selected antidepressants in human breast milk as it was free from matrix effects which were often encountered in breast milk drug analysis [43].In HPLC analysis, thermal desorption at high temperature creates practical problems such as degradation of the polymer, and furthermore, many nonvolatile compounds cannot be completely desorbed from a fiber. Solvent desorption is thus proposed as an alternative method for SPME-HPLC coupling. An organic solvent (static desorption) or the mobile phase (dynamic desorption) is used to desorb the analytes from the SPME fiber. A high throughput SPME-HPLC-UV method has been developed with high sensitivity, precision, and accuracy and enabled the quantification of CIT, SER, PAR, DLX, and FLX in human plasma, following oral administration. For the extraction of 250 mL of plasma, 4 mL of phosphate buffer (pH 7) was added, and the extraction was carried out at 25°C for 40 min, using a modified fibre obtained by the electrochemical deposition (cyclic voltammetry) of a polypyrrole (PPY) film on a stainless steel wire. The drug liquid desorption was made on mobile phase at 25°C for 15 min. The LOD was found 20 ng/mL for CIT and PAR and 25 ng/mL for FLX and SER, while 16 ng/mL for DLX [44].At times,  a fully automated procedure was also used and allowed online extraction of the drug by HPLC. CIT and its S-enantiomer escitalopram (EsCIT) were quantitatively determined from serum by column-switching HPLC in combination with UV and obtained LOD = 6 ng/mL. The method was found to be sensitive and time and solvent saving [45]. A highly selective HPLC method has been developed to detect secondary amine-bearing antidepressants (PAR and FLX) and their metabolites by using 7,7,8,8 tetracyanoquinodimethane (TCNQ) as a derivatizing agent in plasma samples. The method was based on purple chromogens formed by the displacement reaction of the drugs with a derivatizing agent. The resulting chromogens were directly separated by RP-HPLC. For chromatographic separation, C18 column was used in combination with acetonitrile-water (60 : 40) as the mobile phase and achieved complete separation in 40 min. The LOD observation varied mostly between 240 and 480 ng/mL [46].


### 2.1.3. Fluorescence Detector

It has also been observed that the fluorescence detection has some advantages over ultraviolet detection. In addition to higher sensitivity, FD is more specific and selective in reducing some interfering peaks. It was the first detection method used for the analysis of CIT and its DCIT. This method required the LLE extraction of the analytes with hexane from alkaline plasma and their derivatization with 9,10-dimethoxyanthracene-2-sulfonate [47]. Similar detection technique has been applied for the detection of CIT and its metabolites in plasma samples with a successive column-switching technique. Plasma samples were injected directly onto a guard column where the analytes were retained and later eluted, employing a six-port valve by the backflush method. The compounds were analyzed at an excitation wavelength of 249 and an emission wavelength of 302 nm [48].SBSE, coupled with HPLC-FD, was used for the quantification of FLX, CIT, and VLF and their active metabolites NFLX, DCIT, DDCIT, and O-DVLF in plasma, urine, and brain tissue samples. For each matrix, the analytical method has been assessed by studying the linearity and the intra- and interday accuracy (89–113%) and precision (RSD < 13%). In addition to this, the improvement in quantification limits was observed which made it as an applicable procedure for all the matrices useful during the forensic analysis. The use of SBSE favours improvement in the LOQ, as the surface of extraction is higher than microextraction fibers (Figure3) [49].Figure 3
Chromatograms obtained from plasma and brain tissue of rat and urine of human.The retention time of the compounds was Fluoxetine (FLX) = 21.2 min and its metabolite norfluoxetine (NFLX) = 16.1 min, Citalopram (CIT) = 10.6 min and its metabolites o-desmethylvenlafaxine (ODV) = 3.1 min, didesmethylcitalopram (DDCIT) = 7.0 min, venlafaxine (VLF) = 7.8 min, and desmethylcitalopram (DCIT) = 8.6 min. (Reproduced with the permission from [49])Likewise, a quantitative analysis of PAR and MCP has been achieved using an isocratic RP-HPLC combined with FD from human serum samples which reported good linearity and correlation coefficients (r=0.999). Extraction was done with LLE which gave high efficiency, selectivity, and simplicity for the given method and observed less elution time for PAR (7.2 min) [50]. This is very low as compared to the above reported procedure (15.3 min) in spite of the use of high throughput SPE [42].Similarly, FLX was detected in serum using 4-chloro-7-nitro-2,1,3-benzofurazan as a precolumn derivatizing agent in order to improve the sensitivity of the assay. The chromatographic separation was performed on C18 column, and the fluorescence derivatives of the drugs were monitored at the excitation and emission wavelengths of 470 and 537 nm, respectively. Achieved LOQ (0.5 ng/mL) within 6 min, which was more satisfactory than other reported methods, which allowed the determination of low blood FLX levels also. It has been observed that the formations of derivative products were highly dependent on the pH of buffer solution, time, temperature, concentration of the labelling agent, and polarity of the medium. [51]. Other derivatizing agents have also been used to improve the sensitivity of the assay as 4-fluoro-7-nitrobenzofurazan [52], fluorescamine [53], and dansyl chloride [54]. However, while using fluorescamine and dansyl derivatives, there might be a possibility of poor photo stability and band broadening of the resulted peaks. Table 1 represents the LC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.Table 1
Liquid chromatographic methods developed for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction method
Detector system
Sensitivity
References
Mass detector
Plasma
SER andN-DSER
LLE
TMS
LOQ = 0.5 ng/mL
[21]
Plasma
FLX and NFLX
LLE
ESI, SIM*
LOQ = 2.5μg/mL for FLX and 10 μg/mL NFLX
[25]
Plasma
FLX
SBSE
ESI, SIM
LOQ = 30 pg/mL (liquid desorption) and 1.37 pg/mL (thermal desorption)
[26]
Hair
CIT, DCIT, and DDCIT
LLE
TMS
LOD = 10 pg/mgLOQ = 20 pg/mg
[33]
Plasma
VLF,O-DVLF, N-DVLFN,O-didesmethylvenlafaxine (DDVLF)
LLE
MS
LOD = 0.4, 0.2, 0.3, and 0.2 ng/mL andLLOQ = 3.5, 2.2, 2.7, and 1.9 ng/mL for VLF, O-DVLF, N-DVLF, and DDVLF, respectively
[75]
Plasma
SER
SPE
Triple stage(ESI, SRM)
LOQ = 0.5 ng/mL
[76]
Oral fluid and plasma
FLX, NFLX PAR, SER, FLV, CIT, and VLF
Automated SPE
TMS (ESI), with triple quadrupole
LOQ = 2 ng/mL in oral fluid, 2 ng/mL in plasma (except FLV 10 ng/mL and NFLX 4 ng/mL)
[77]
Plasma
FLX and NFLX
Automated SPE
Triple stage(ESI, SRM)
LLOQ = 0.5 ng/mL
[78]
Plasma
FLX and NFLX
Online extraction using column switching
ESI, SIM
LOQ = 25 ng/mL
[79]
Plasma
VLF andO-DVLF
SPE
TMS, SRM
LOQ = 3 ng/mL for VLF, 6 ng/mL for DVLF
[80]
Plasma
CIT
LLE
ESI, SIM
LOQ = 0.50 ng/mL
[81]
Plasma
CIT and DCIT
Deproteinization
MS (SIM)
LOQ = 0.25 to 0.5 ng/mL
[82]
Plasma
FLV, PAR, and MCP
SPE
QMS-(SSI)*
LOD = 0.05 to 0.13μg/mL and LOQ = 0.10 to 0.20 μg/mL
[83]
Plasma
FLX and NFLX
LLE
TMS (ESI)
LOQ = 0.15 ng/mL
[84]
Plasma
FLX, CIT, PAR, and VLF
SPE
MS (ESI)
LOD, FLX = 0.5, CIT = 0.3, PAR = 0.3, and VEN = 0.1 ng/mL
[85]
Blood
FLX, PAR, SER, FLV, CIT, VLF, NFLX, DCIT, DDCIT, and DVLF
LLE
TMS
LOD = 5–500 ng/mL and LOQ = 5 ng/mLLOD = 20–2000 ng/mL for VLF and DVLF LOQ = 20 ng/mL for VLF and DVLF
[86]
UV detector
Urine and plasma
SER
HF-LPME
UV
LOD = 0.5μg/L LOQ = 2.3 μg/L
[18]
Plasma
CIT and DCIT
LLE
UV
LOD = 5 ng/mL
[38]
Plasma
CIT, SER, FLX, PAR, and DLX
SBSE
UV
LOD = 20 ng/mL for CIT and PAR, 25 ng/mL for FLX and SER and 16 ng/mL for DLX
[44]
Plasma
FLX, SER, PAR, and CIT
LLE
UV
LOQ = 10 ng/mL for FLX, SER, and PAR and 5 ng/mL for CIT
[87]
Plasma and urine
FLX, PAR, and VLF
SPE
UV
LOD = 0.2–0.6 ng/μL in plasma and 0.1–0.8 ng/μL in urine
[88]
Plasma
CIT, FLX, PAR
SPE
UV
LOQ = 0.05 to 0.26 mM
[89]
Plasma
CIT, DCIT, DDCIT, FLV, PAR, SER,FLX, NFLX, VLF, andO-DVLF
LLE
UV
LOQ = 25 ng/mL,   100 ng/mL for VLF andO-DVLF
[90]
Plasma
R- and S-CIT, R- and S-DCIT, and R- and S-DDCIT
LLE
UV
LOD > 2 ng/mL
[91]
Fluorescence detector
Plasma
FLX and NFLX
LLE
FD
LLOD = 3 mg/L
[17]
Plasma
SER and DSER
SPE
FD
LOQ = 1.0 ng/mL  and LOD = 0.3 ng/mL
[22]
Plasma
CIT, DCIT, DDCIT
SPE
FD
LOQ = 1.5 to 2 ng/mL
[32]
Plasma, urine, and brain tissue
CIT, DCIT, DDCIT, FLX, NFLX, VLF, andO-DVLF
SBSE
FD
LOQ = 0.2 to 2 mg/L in plasma, 2 to 20 ng/g in brain tissue, and 1 to 10 mg/L in urine
[49]
Serum
SER
LLE
FD
LOQ up to 2 ng/mL
[53]
Plasma
CIT, DCIT, DDCIT, PAR, and PAR metabolites
SPE
FD
LOQ = 0.025 to 0.12 mmol/L
[92]
Serum
CIT, FLX, and NFLX
LLE
FD
LOQ = 50 mg/L
[93]
Plasma
FLX and NFLX
HF-LPME
FD
LOQ = 5 ng/mL
[89]
Plasma
CIT, DCIT, and DDCIT
SPE
FD
LOD = 0.8 nmol/L
[94]
Diode array detector
Plasma
FLX, NFLX, SER, PAR, CIT, FLV, VLF, MCP, and DVLF
LLE
DAD
LOD = 2.5 to 5 ng/mL
[95]
Urine
CIT, DCIT, DDCIT, FLX, and NFLX
SPME
DAD
LOD = 0.01–0.09 mg/L
[96]
Abbreviations*: SIM: Single Ion Monitoring; QMS: Quadrupole Mass Spectrometry; SSI: Sonic Spray Ionisation.


## 2.1.1. Mass Detector

LC coupled with single-stage (LC-MS) or tandem mass spectrometry (TMS or LC-MS-MS) is becoming increasingly important in routine laboratory analysis of antidepressant drugs especially from whole blood, plasma/serum, and urine. The alternative matrices such as hair and oral fluid have also given satisfactory results [20]. Moreover, MS has been the most commonly used detector in the determination of antidepressants, the high specificity and the lower limits of detection obtained being the main advantages with this detection technique. With a mass spectrometer, it is possible to monitor only the mass of the analyte which is extremely beneficial in the structural elucidation studies. A precise, sensitive, and high throughput LC-TMS method was developed for simultaneous determination of SER and its primary metabolite, N-desmethylsertraline (DSER) in human plasma. The analytes were extracted from plasma via liquid-liquid extraction (LLE) in methyl tert-butyl ether. The chromatographic conditions were aimed for getting an adequate response, a sharp peak shape, and a short run time per analysis. It was observed that 1 M (2.5 mL) ammonium trifluoroacetate in 750 mL methanol + 250 mL deionized water as the mobile phase provides faster elution, better efficiency, and a peak shape. The use of Betasil C8 chromatographic column helped in the separation and elution of both compounds in a very short time of 1.40 and 1.47 min for SER and N-DSER, respectively. The maximum on-column loading per sample injection was 0.45 ng for both analytes [21]. This volume was considerably less as compared to other reported procedures (injection volume of 20 μL), but the former method helped in maintaining the efficiency and lifetime of the column which in turn gives consistent and reproducible recoveries of analytes [22].Quantitative estimations of VLF and desmethylvenlafaxine (DVLF) were carried out in biological samples obtained from postmortem cases. VLF and one of its active metabolites, DVLF, were identified by LC-MS using atmospheric pressure ionisation (API) electrospray in positive mode, followed by extraction throughn-butyl chloride. Chromatographic separation was performed on C18 column and the mobile phase consisted of ammonia (0.05 M/methanol/tetrahydrofuran (32.5 : 67.0 : 0.5 vol%)) titrated to pH = 10.0 with 12 M formic acid and achieved retention time of 9.1 and 3.5 min for VLF and DVLF, respectively. Moreover, the postmortem tissue concentrations observed were 0.1–36 and <0.05–3.5 mg/L (peripheral blood), <0.05–22 and <0.05–9.9 mg/kg (liver), and <0.05–10 and <0.05–1.5 mg/L (vitreous), <0.05–55 and <0.05–21 mg/L (urine), respectively. Additionally, it was also reported that VLF alone was not responsible for intoxication in the above described postmortem studies. However, it has shown synergism in presence of other drugs like FLX and moclobemide, which could be liable for fatality [23]. An atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) method was developed for the identification and quantitation of CIT, FLV, and PAR in human plasma. Direct analysis of plasma samples was done after dilution in 0.1% formic acid, followed by direct injection into the HPLC system, avoiding a time-consuming sample preparation step. Proteins and other large biomolecules were removed during an online sample cleanup step. The limit of quantitation (LOQ) obtained was below 20 ng/mL which permitted the direct analysis of plasma samples [24].The method of sample introduction to the ionisation source is very important while using a mass detector, and it often depends on the ionisation method being used. The ionisation method to be used should depend on the type of sample under investigation. Different types of ionisation sources were used for quantification of SSRIs and SNRIs. Electron spray ionisation (ESI) mode, in combination with LC, was used for quantification of FLX in plasma samples [25, 26]. As compared to the above electron spray ionisation (ESI) method, APCI mode has also provided higher mass spectrometric response and less susceptibility to ion suppression [27].Fully automated online solid phase extraction (SPE) coupled to LC-TMS was developed and validated for direct analysis of 14 antidepressants including SSRIs and their metabolites in plasma. The integration of sample extraction and HPLC separation into a single system permitted direct injection of the plasma without prior sample pretreatment. An automated online SPE-LC-MS/MS method permitted direct injection of 50 mL of plasma without prior sample pretreatment, ensuring the analysis of the samples in 20 min. Several deuterated drugs were employed as internal standards. For the detection, a tandem mass spectrometer with a triple quadrupole was employed operating in electrospray in the positive ionization mode. Selectivity of the method was achieved by a combination of the retention time and two precursor-product ion transitions for the nondeuterated compounds. The LOQs were estimated as 10 ng/mL for all compounds [28].Recently, a new kind of sample preparation media has been developed as restricted access media (RAM) and possesses a biocompatible surface and adsorption centres which provide very fine extraction in minimal amount of sampling (nearly 1μL). This could be related to miniaturization of chromatographic separation, especially with the improvement of mass sensitivity. By giving minimal sample pretreatment through RAM, FLX was detected in plasma by direct analysis through LC-MS in total run time of 8 min. Calibration curves are constructed under a linear range of 1–250 ng/mL with LOQ = 1 ng/mL. This demonstrates the applicability of a new capillary RAM-LC-MS/MS approach for the detection of antidepressant drugs with less consumption of time, solvent, and samples [29]. Likewise, the integration of the sample extraction apparatus with LC separation and MS detection into a single system permitted the direct injection of diluted urine or plasma samples after filtration. An automated in-tube solid-phase microextraction (SPME) LC-MS method was developed for the analysis of SSRIs and SNRIs in urine and plasma. A hybrid organic-inorganic silica monolith with cyano-ethyl functional groups was prepared and used as a sorbent for in-tube SPME. The total process time was 30 min, and only 30 μL of urine or plasma was required in one analysis cycle, achieving good extraction efficiencies and recoveries (urine and plasma were from 75.2% to 113.0%) with no matrix interferences. Limit of detection (LOD) was found as 0.06–2.84 ng/mL in urine and 0.07–2.95 ng/mL in plasma. This could minimize repetitive work and also eliminates the analyst’s exposure to hazardous and toxic solvents [30].A new type of polymer column (Shim-pack MAYI-ODS) has been used for the separation of SER, FLV, and PAR in human serum by LC-MS/MS. This column is having a unique online dilution by pass channel which can reduce labour and enhances efficiency. The recoveries of the three drugs in human serum were observed as 29.2%–45.7% at 20 ng/mL and 52.0%–53.7% at 80 ng/mL of injection, and each drug had a detection limit of 1–3 ng/mL. Thus, the method enabled direct injection of crude biological samples without complicated pretreatments and without column switching system [31].CIT and its metabolites desmethylcitalopram (DCIT) and didesmethylcitalopram (DDCIT) were separated from plasma samples through SPE, using two types of cartridges during extraction, C8 and Oasis hydrophilic-lipophilic balance cartridges. C8 cartridge has produced good extraction yields, but some interference was also detected due to plasma matrix, while the results obtained with Oasis hydrophilic-lipophilic balance cartridges were better in terms of extraction yield and selectivity. Thus, the later procedure was chosen for extraction, achieving LOQ = 1.5 ng/mL for CIT and DCIT and 2.0 ng/mL for DDCIT [32]. CIT and DCIT were also quantified from neonatal hair samples which suggest gestational exposure of these drugs. A LC-TMS method has been developed and validated for the assay of CIT in neonatal hair samples which also explores the utility of alternative test matrices in detection [33].On the contrary, a simple chemical treatment to the biological sample can also allow the direct injection of the supernatant to the column. A very sensitive HPLC-TMS with ESI method was developed for the simultaneous determination of FLX and norfluoxetine (NFLX) in human plasma. It was observed that the addition of formic acid to mobile phase could improve sensitivity by promoting the ionization of analytes. Acetonitrile was found to be more favourable for the chromatographic separation of FLX and NFLX from matrix than methanol and able to produce high sensitivity (retention time = 3.8 min), lowest background noise, and most stable MS signals (Figures2(a) and 2(b)) [34].(a) The product ion spectra of [M + H]+ of (A) Fluoxetine and (B) Norfluoxetine. (b) Chromatogram of plasma sample spiked with (A) Fluoxetine (3.0 ng/mL) and (B) Norfluoxetine (2.6 ng/mL) (reproduced with the permission from [34]).
(a)
(b)Except for all these proposed methods, a very noble HPLC-TMS method (with single sample/triple injection approach) was developed and validated for the simultaneous determination of forty-eight antidepressants (including SSRIs and SNRIs), antipsychotics, and their pharmacologically active metabolites in serum. The chromatographic separation was performed on a monolithic C18 column (50 mm × 4.6 mm) with methanol gradient and 5 mM acetate buffer (pH 3.9). The sample volume needed for analysis was very less (0.1 mL) which requires only protein precipitation and stepwise dilution for sample preparation. After ESI, positive ion fragments were detected in the multiple reaction monitoring mode. The LOQ was found as 1.0, 2.17, 1.17, 1.07, 0.70 ng/mL for CIT, FLX, FLV, PAR, and SER, respectively. The advantage of the method was to minimize the expenditure for routine determination of large set of individuals with varying drug combinations [35].Newer techniques like ultra-high performance liquid chromatography (UHPLC) have managed to reduce the time analysis and the effectiveness of the separations. An UHPLC-time-of-flight (UHPLC-TOF-MS) method has been proposed for the simultaneous screening and quantification of 52 drugs, including SSRIs in 15 autopsy hair samples, with a total chromatographic run time of 17 min. Hair samples were liquid-liquid extracted with methanol, acetonitrile, ammonium formate (2 mM, 8% acetonitrile, pH and 5.3) overnight at 37°C. The LODs for CIT, FLX, and VLF were found to be 0.01 ng/mL, and for SER and PAR they were found to be 0.02 and 0.05 ng/mL, respectively [36]. Likewise, HPLC-HRMS (high resolution mass spectroscopy) method for simultaneous screening and quantification of 28 drugs, including VLF and FLX, was developed and validated on hair samples. The samples (2.5 mg) were extracted with 200 μL of a mixture of water: acetonitrile: 1 M trifluoroacetic acid (80 : 10 : 10, v/v). Total chromatographic run time was 26 min (VLF = 16.3 min and FLX = 20.5 min), reaching lower limit of quantitation (LLOQs) as low as 0.1–0.5 ng/mg, which are particularly suitable for the forensic determination of selected antidepressants [37].


## 2.1.2. UV Detector

Owing to the wide applicability of the UV detector, it has been deemed suitable for the development of multiresidue liquid chromatography methods [38]. In order to achieve good chromatographic separation and enhancement of signals, different types of extraction procedures were adopted for the removal of interfering particles such as LLE, SPE, stir bar sorptive extraction (SBSE), and liquid-phase microextraction (LPME) which has given a detection limit up to nano or pico level [39]. Sometimes for multicomponent analysis [40] dual UV wavelength detection [41] has also been used for the identification of compounds. An isocratic reverse phase HPLC (RP-HPLC) method with UV detection was optimised for the determination of SER, FLV, FLX, PAR, CIT, MCP, VLF, and o-desmethylvenlafaxine (ODV) in human serum. The SPE, yielded reproducible recoveries from 75 to 99%, allowed efficient and rapid analysis in minimum amount of time [42].Recently, a newer kind of hollow fibre based liquid-phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of SER from urine and plasma samples prior to its determination by HPLC-UV. This is a kind of miniaturised isolation technique based on the use of disposable, porous, hollow fibres made of polypropylene, which can combine extraction, concentration, and sample cleanup in one step only. The calibration curves were obtained in the range of 5–500μg/L with reasonable linearity (R2 > 0.998). Hence, the method indicates the applicability of hollow fibre microextraction as an excellent cleanup procedure and can be served as a simple and sensitive method for monitoring antidepressant drugs in the biological samples [18].Consequently, there has been an increased effort to develop standardized methods for quantifying psychotropic medications in breast milk which also shows the in utero exposure of these drugs. A method was developed on HPLC-UV for measuring antidepressants in human breast milk. The extraction of drugs was achieved by both techniques as LLE and SPE. Three kinds of assays were performed during analysis, in which assay 1 measured FLX, NFLX, and FLV; assay 2 measured SER and DSER; and assay 3 measured the tricyclic antidepressants. This method was found as highly accurate and precise in measuring the selected antidepressants in human breast milk as it was free from matrix effects which were often encountered in breast milk drug analysis [43].In HPLC analysis, thermal desorption at high temperature creates practical problems such as degradation of the polymer, and furthermore, many nonvolatile compounds cannot be completely desorbed from a fiber. Solvent desorption is thus proposed as an alternative method for SPME-HPLC coupling. An organic solvent (static desorption) or the mobile phase (dynamic desorption) is used to desorb the analytes from the SPME fiber. A high throughput SPME-HPLC-UV method has been developed with high sensitivity, precision, and accuracy and enabled the quantification of CIT, SER, PAR, DLX, and FLX in human plasma, following oral administration. For the extraction of 250 mL of plasma, 4 mL of phosphate buffer (pH 7) was added, and the extraction was carried out at 25°C for 40 min, using a modified fibre obtained by the electrochemical deposition (cyclic voltammetry) of a polypyrrole (PPY) film on a stainless steel wire. The drug liquid desorption was made on mobile phase at 25°C for 15 min. The LOD was found 20 ng/mL for CIT and PAR and 25 ng/mL for FLX and SER, while 16 ng/mL for DLX [44].At times,  a fully automated procedure was also used and allowed online extraction of the drug by HPLC. CIT and its S-enantiomer escitalopram (EsCIT) were quantitatively determined from serum by column-switching HPLC in combination with UV and obtained LOD = 6 ng/mL. The method was found to be sensitive and time and solvent saving [45]. A highly selective HPLC method has been developed to detect secondary amine-bearing antidepressants (PAR and FLX) and their metabolites by using 7,7,8,8 tetracyanoquinodimethane (TCNQ) as a derivatizing agent in plasma samples. The method was based on purple chromogens formed by the displacement reaction of the drugs with a derivatizing agent. The resulting chromogens were directly separated by RP-HPLC. For chromatographic separation, C18 column was used in combination with acetonitrile-water (60 : 40) as the mobile phase and achieved complete separation in 40 min. The LOD observation varied mostly between 240 and 480 ng/mL [46].


## 2.1.3. Fluorescence Detector

It has also been observed that the fluorescence detection has some advantages over ultraviolet detection. In addition to higher sensitivity, FD is more specific and selective in reducing some interfering peaks. It was the first detection method used for the analysis of CIT and its DCIT. This method required the LLE extraction of the analytes with hexane from alkaline plasma and their derivatization with 9,10-dimethoxyanthracene-2-sulfonate [47]. Similar detection technique has been applied for the detection of CIT and its metabolites in plasma samples with a successive column-switching technique. Plasma samples were injected directly onto a guard column where the analytes were retained and later eluted, employing a six-port valve by the backflush method. The compounds were analyzed at an excitation wavelength of 249 and an emission wavelength of 302 nm [48].SBSE, coupled with HPLC-FD, was used for the quantification of FLX, CIT, and VLF and their active metabolites NFLX, DCIT, DDCIT, and O-DVLF in plasma, urine, and brain tissue samples. For each matrix, the analytical method has been assessed by studying the linearity and the intra- and interday accuracy (89–113%) and precision (RSD < 13%). In addition to this, the improvement in quantification limits was observed which made it as an applicable procedure for all the matrices useful during the forensic analysis. The use of SBSE favours improvement in the LOQ, as the surface of extraction is higher than microextraction fibers (Figure3) [49].Figure 3
Chromatograms obtained from plasma and brain tissue of rat and urine of human.The retention time of the compounds was Fluoxetine (FLX) = 21.2 min and its metabolite norfluoxetine (NFLX) = 16.1 min, Citalopram (CIT) = 10.6 min and its metabolites o-desmethylvenlafaxine (ODV) = 3.1 min, didesmethylcitalopram (DDCIT) = 7.0 min, venlafaxine (VLF) = 7.8 min, and desmethylcitalopram (DCIT) = 8.6 min. (Reproduced with the permission from [49])Likewise, a quantitative analysis of PAR and MCP has been achieved using an isocratic RP-HPLC combined with FD from human serum samples which reported good linearity and correlation coefficients (r=0.999). Extraction was done with LLE which gave high efficiency, selectivity, and simplicity for the given method and observed less elution time for PAR (7.2 min) [50]. This is very low as compared to the above reported procedure (15.3 min) in spite of the use of high throughput SPE [42].Similarly, FLX was detected in serum using 4-chloro-7-nitro-2,1,3-benzofurazan as a precolumn derivatizing agent in order to improve the sensitivity of the assay. The chromatographic separation was performed on C18 column, and the fluorescence derivatives of the drugs were monitored at the excitation and emission wavelengths of 470 and 537 nm, respectively. Achieved LOQ (0.5 ng/mL) within 6 min, which was more satisfactory than other reported methods, which allowed the determination of low blood FLX levels also. It has been observed that the formations of derivative products were highly dependent on the pH of buffer solution, time, temperature, concentration of the labelling agent, and polarity of the medium. [51]. Other derivatizing agents have also been used to improve the sensitivity of the assay as 4-fluoro-7-nitrobenzofurazan [52], fluorescamine [53], and dansyl chloride [54]. However, while using fluorescamine and dansyl derivatives, there might be a possibility of poor photo stability and band broadening of the resulted peaks. Table 1 represents the LC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.Table 1
Liquid chromatographic methods developed for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction method
Detector system
Sensitivity
References
Mass detector
Plasma
SER andN-DSER
LLE
TMS
LOQ = 0.5 ng/mL
[21]
Plasma
FLX and NFLX
LLE
ESI, SIM*
LOQ = 2.5μg/mL for FLX and 10 μg/mL NFLX
[25]
Plasma
FLX
SBSE
ESI, SIM
LOQ = 30 pg/mL (liquid desorption) and 1.37 pg/mL (thermal desorption)
[26]
Hair
CIT, DCIT, and DDCIT
LLE
TMS
LOD = 10 pg/mgLOQ = 20 pg/mg
[33]
Plasma
VLF,O-DVLF, N-DVLFN,O-didesmethylvenlafaxine (DDVLF)
LLE
MS
LOD = 0.4, 0.2, 0.3, and 0.2 ng/mL andLLOQ = 3.5, 2.2, 2.7, and 1.9 ng/mL for VLF, O-DVLF, N-DVLF, and DDVLF, respectively
[75]
Plasma
SER
SPE
Triple stage(ESI, SRM)
LOQ = 0.5 ng/mL
[76]
Oral fluid and plasma
FLX, NFLX PAR, SER, FLV, CIT, and VLF
Automated SPE
TMS (ESI), with triple quadrupole
LOQ = 2 ng/mL in oral fluid, 2 ng/mL in plasma (except FLV 10 ng/mL and NFLX 4 ng/mL)
[77]
Plasma
FLX and NFLX
Automated SPE
Triple stage(ESI, SRM)
LLOQ = 0.5 ng/mL
[78]
Plasma
FLX and NFLX
Online extraction using column switching
ESI, SIM
LOQ = 25 ng/mL
[79]
Plasma
VLF andO-DVLF
SPE
TMS, SRM
LOQ = 3 ng/mL for VLF, 6 ng/mL for DVLF
[80]
Plasma
CIT
LLE
ESI, SIM
LOQ = 0.50 ng/mL
[81]
Plasma
CIT and DCIT
Deproteinization
MS (SIM)
LOQ = 0.25 to 0.5 ng/mL
[82]
Plasma
FLV, PAR, and MCP
SPE
QMS-(SSI)*
LOD = 0.05 to 0.13μg/mL and LOQ = 0.10 to 0.20 μg/mL
[83]
Plasma
FLX and NFLX
LLE
TMS (ESI)
LOQ = 0.15 ng/mL
[84]
Plasma
FLX, CIT, PAR, and VLF
SPE
MS (ESI)
LOD, FLX = 0.5, CIT = 0.3, PAR = 0.3, and VEN = 0.1 ng/mL
[85]
Blood
FLX, PAR, SER, FLV, CIT, VLF, NFLX, DCIT, DDCIT, and DVLF
LLE
TMS
LOD = 5–500 ng/mL and LOQ = 5 ng/mLLOD = 20–2000 ng/mL for VLF and DVLF LOQ = 20 ng/mL for VLF and DVLF
[86]
UV detector
Urine and plasma
SER
HF-LPME
UV
LOD = 0.5μg/L LOQ = 2.3 μg/L
[18]
Plasma
CIT and DCIT
LLE
UV
LOD = 5 ng/mL
[38]
Plasma
CIT, SER, FLX, PAR, and DLX
SBSE
UV
LOD = 20 ng/mL for CIT and PAR, 25 ng/mL for FLX and SER and 16 ng/mL for DLX
[44]
Plasma
FLX, SER, PAR, and CIT
LLE
UV
LOQ = 10 ng/mL for FLX, SER, and PAR and 5 ng/mL for CIT
[87]
Plasma and urine
FLX, PAR, and VLF
SPE
UV
LOD = 0.2–0.6 ng/μL in plasma and 0.1–0.8 ng/μL in urine
[88]
Plasma
CIT, FLX, PAR
SPE
UV
LOQ = 0.05 to 0.26 mM
[89]
Plasma
CIT, DCIT, DDCIT, FLV, PAR, SER,FLX, NFLX, VLF, andO-DVLF
LLE
UV
LOQ = 25 ng/mL,   100 ng/mL for VLF andO-DVLF
[90]
Plasma
R- and S-CIT, R- and S-DCIT, and R- and S-DDCIT
LLE
UV
LOD > 2 ng/mL
[91]
Fluorescence detector
Plasma
FLX and NFLX
LLE
FD
LLOD = 3 mg/L
[17]
Plasma
SER and DSER
SPE
FD
LOQ = 1.0 ng/mL  and LOD = 0.3 ng/mL
[22]
Plasma
CIT, DCIT, DDCIT
SPE
FD
LOQ = 1.5 to 2 ng/mL
[32]
Plasma, urine, and brain tissue
CIT, DCIT, DDCIT, FLX, NFLX, VLF, andO-DVLF
SBSE
FD
LOQ = 0.2 to 2 mg/L in plasma, 2 to 20 ng/g in brain tissue, and 1 to 10 mg/L in urine
[49]
Serum
SER
LLE
FD
LOQ up to 2 ng/mL
[53]
Plasma
CIT, DCIT, DDCIT, PAR, and PAR metabolites
SPE
FD
LOQ = 0.025 to 0.12 mmol/L
[92]
Serum
CIT, FLX, and NFLX
LLE
FD
LOQ = 50 mg/L
[93]
Plasma
FLX and NFLX
HF-LPME
FD
LOQ = 5 ng/mL
[89]
Plasma
CIT, DCIT, and DDCIT
SPE
FD
LOD = 0.8 nmol/L
[94]
Diode array detector
Plasma
FLX, NFLX, SER, PAR, CIT, FLV, VLF, MCP, and DVLF
LLE
DAD
LOD = 2.5 to 5 ng/mL
[95]
Urine
CIT, DCIT, DDCIT, FLX, and NFLX
SPME
DAD
LOD = 0.01–0.09 mg/L
[96]
Abbreviations*: SIM: Single Ion Monitoring; QMS: Quadrupole Mass Spectrometry; SSI: Sonic Spray Ionisation.


## 2.2. Gas Chromatography (GC)

The use of GC with varying detectors has proved to be a powerful tool in the area of underivatized and derivatized drug analysis from biological extracts. The following are the different detectors used in combination with gas chromatography for the analysis of SSRIs and SNRIs in a biological matrix.


### 2.2.1. Mass Detector (MS)

A method was developed based on a capillary gas chromatography with mass spectrometry detection in selected ion monitoring (SIM) mode for the analysis of CIT, FLX, DCIT, DDCIT, and NFLX in urine samples. An optimized SPE procedure was applied which provided a preconcentration factor of 10, and no derivatization step was required [55].In order to achieve a high degree of separation for SER, PAR, CIT, DCIT, DDCIT, and DSER in a plasma sample, reextraction was done at varying pH, resulting in less interference from endogenous compounds. Such kinds of GC separations were performed using a cross-linked 30 m DB-35 ms silica capillary column, and helium (He) was used as a carrier gas (flow rate = 1.5 mL/min), with a mass spectroscopic measurement in ESI mode. The simultaneous chromatographic separation was achieved after the derivatization of analytes with reagent N-methyl-bis(trifluoroacetamide) [56]. SPME-GC has been used for the determination of SSRIs in urine samples. Fibers of 65 mm polydimethylsiloxane-divinylbenzene (PDMS-DVB) were used for the determination of the target SSRIs after simple in situ derivatization by acetylation of FLV, FLX, and SER. A 1.5 g aliquot of sodium chloride was then added to the samples. The vial was sealed with an aluminum cap and a teflon faced septum. It was then immersed in a water bath at 100°C and left to equilibrate for 5 min. To perform the extraction, the SPME fiber was exposed to the magnetically stirred sample for 30 min. The fiber was then immediately inserted into GC injection port and analysis was performed. Desorption time was set at 3 min. The time required for SPME step and for GC analysis (30 min each) enabled high throughputness [57].Derivatization is an important process which helps in large-scale screening of varieties of drug. This helps in the conversion of thermally labile and nonvolatile analytes to a suitable form for GC analysis, which has extended its use in the detection of antidepressant drugs and their metabolites. The separation of VLF, FLV, FLX, and CIT in blood samples was obtained by GC-MS operating in select ion monitoring (SIM) mode. Analytes were isolated by high yield LLE followed by derivatization throughtert-butyldimethylsilylation (MTBSTFA) and tri-methylsilylation (MSTFA) reagents. Thus, the analytes were analysed in tert-butyldimethylsilylated (TBDMS) and tri-methylsilylated forms (TMS) by GC-MS. Due to superior derivatization power and intensive derivatives, silylation was believed to have the best properties for large-scale screening with a variety of substances having structural differences. The method is feasible for highly sensitive, reliable, and routinely performed forensic toxicological analysis that produced satisfactory results with different concentration ranges of analytes in small sample volume [58]. In general, the derivatizing agents mostly preferred during GC separation of antidepressant drugs were N-methyl-N-trimethylsilyl-trifluoroacetamide for silylation, acetic anhydride-pyridine for acetylation, and trimethylammonium hydroxide for methylation [59].The identification and quantification of psychoactive drugs like antidepressants can be obtained using alternative biological matrices like oral fluid. Analyses of SER and PAR were obtained with GC-MS(-MS), operating in SIM mode after their SPE from oral fluid. Due to the basic nature of these drugs, eluting solvent used in SPE is a mixture of dichloromethane: isopropanol: ammonium (80 : 20 : 2, v/v/v). The chromatographic separation was performed on methylsilicone capillary column, and analytes were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and obtained retention time of 12.16 (SER) and 12.49 min (PAR). The method showed the applicability of alternative test matrices like oral fluid which have been extensively and successfully used to access acute and chronic exposure of drugs of abuse [60]. Similarly, in a suicidal case of multiple drug ingestion, CIT was identified and quantified in the postmortem samples by GC-MS. The concentrations of CIT found in femoral blood, heart blood, and urine were 0.88 mg/L, 1.16 mg/L, and 0.9 mg/L, respectively. This method also shows its applicability in the determination of CIT in postmortem tissue samples like liver, brain, gastric content, and so forth [61].Moreover, full-scan EI ionization mode is also considered as the reference technique for drug screening procedures since it plays an important role in the analysis of drugs in forensic toxicology and doping control. The main advantage of this technique is that it provides comparable GC-MS spectra on all commercially available instruments since the standardized ionization EI mode produces unique and uniform mass fragmentation patterns. As a consequence, large mass spectral libraries have been constructed which enable the identification of unknown compounds [62]. Table 2 represents the GC methods that appeared in the literature for the determination of new generation antidepressant drugs and their metabolites.Table 2
Gas chromatographic methods developed on for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction       method
Detector system
Sensitivity
References
Mass detector
Urine
FLX, VLF, SER, and CIT
SPE
EI-MS
LOD, 2, 0.02, 1.25,  and 10μg/mL for FLX, VLF, SER, and CIT
[16]
Plasma
CIT, DCIT, DDCIT, FLX, and NFLX
SPE
MS
LOD = 0.7  to 33.6μg/mL
[55]
Plasma
CIT, DCIT, DDCIT, SER, DSER, and PAR
LLE
MS
LOQ = 2 ng/mL for CIT and PAR, 1 ng/mL for SER, 0.5 ng/mL for DSER, DCIT, and DDCIT
[56]
Urine
FLV, FLX, SER, VLF, and CIT
SPME
MS (ESI mode)
LOD < 0.4 ng/mL
[57]
Blood
VLF, FLX, FLV, CIT, and SER
LLE
MS (ESI in SIM mode)
LOD = <10, 25, 25, <10, 4 ng/mL, and LOQ = 25, 50, 25, 25, 50, 25, 20 ng/mL for VLF, FLV, LX, CIT, and SER, respectively
[58]
Oral fluid
SER
SPE
MS in SIM mode.
LOD = 18.6 ng/mL
[60]
Plasma
VLF, CIT, FLX, FLV, SER, PAR, DFLX, DSER, DCIT, and DDCIT
SPE
Mass (ESI and Chemical Ionisation  (PCI*/NCI* source) in SIM mode)
LOQ = 5 to 12.5 ng/mL in ESI and PICI mode and LOQ = 1 to 6.25 ng/mL in NCI mode
[97]
Plasma
CIT, DCIT, and DDCIT
LLE
MS
LOQ = 1 to 2 ng/mL
[98]
Other detectors
Serum
CIT and DCIT
LLE
NPD
LOQ = 10–20 mg/L
[63]
Blood
CIT
SPE
NPD
LOQ = 70 ng/mL
[99]
Urine
CIT, FLX, FLV, SER, and PAR
SPE
FID
LOQ = 91.7,  64.9, 100.3, and 61.4 and 95.1μg/L for CIT, FLX, FLV, SER, and PAR, respectively
[100]
Plasma
CIT, PAR, DLX, FLX, and SER
In-tube SPME
UV
LOQ = 50 to 500 ng/mL
[101]
Abbreviations*: PCI: Positive Chemical Ionization, NCI: Negative Chemical Ionization.


### 2.2.2. Nitrogen Phosphorous Detector (NPD)

Despite the differences in chemical structures of FLX, SER, CIT, VLF, DCIT, DSER, and NFLX,  were successfully assayed in serum samples after LLE, followed by GC-NPD. It has been observed that for GC procedure, polarity of extracting solvent (proportion of isoamyl alcohol in the mixture with hexane) was one of the most important variables influencing extractions. An added factor, which was also considered during analysis, is the nitrogen containing structures of analytes and responsible for the high sensitivity obtained using nitrogen phosphorous detector (NPD) (helps in minimisation of interferences). The method was reported with good linearity (20 to 400 mg/L) and correlation coefficients (r=0.998). The specificity of the method can help during the toxicological screening of antidepressant drugs [63]. Even stereoselective determination of FLX and NFLX enantiomers were obtained by using the two-dimensional capillary gas liquid chromatography with NPD that allows the separation of racemic mixtures of the drugs in plasma or serum. A three-step LLE was used for sample preparation and provided linear calibration between 5–250 ng/mL for (R)- and (S)-FLX and 15–250 ng/mL for (R)- and (S)-NFLX, with LOD = 1.5 ng/mL and 6 ng/mL, respectively [64].A comparative study was done for two types of extraction procedures regarding their recovery, precision, sensitivity, matrix purification efficiency, and less solvent consumption for the detection of SER and VLF in blood samples using GC-NPD. First procedure consists of the employment of chem elut column (diatomaceous earth) based on the principle of solid-liquid absorption extraction and closely related to conventional LLE, while in the second bond, elut certify column was used with mixed SPE having reversed-phase and cation-exchange sorbents. Recoveries of the compounds using chem elut columns at 500 ng/mL were in the range of 28–74% and with LOD and LOQ ranging from 39 to 153 ng/mL and from 128 to 504 ng/mL, respectively, while recoveries of the compounds using bond elut certify columns at 500 ng/mL were in the range of 64–86% and with LODs and LOQs ranging from 21 to 100 ng/mL and from 70 to 330 ng/mL, respectively. Thus, the results suggested that the use of reversed-phase and cation-exchange sorbent bond elut certify columns is more satisfactory for the screening of the selected drugs [65]. In a similar kind of study, the use of mixed-mode bonded-silica bond elut certify columns had shown advantages rather than chem elut columns, in terms of recoveries, cleaner extracts, sensitivity, precision and solvent consumption. It has also been explained that the chem elut column has diatomaceous earth support which was a hydrophilic packing porous material acted as a support for the aqueous phase. Its high surface area ensured that the organic eluents remain immiscible with the aqueous matrix, eliminating emulsion problems and facilitating an efficient interaction between sample and organic solvents, while bond elut certify column was a mixed-mode bonded silica extraction support containing hydrophobic chains and strong cation-exchange moieties. This gave the ability to retain acidic, neutral, basic, and amphoteric drugs on one column which is quite advantageous in the field of forensic toxicology [66]. Table 2 represents the GC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.


## 2.2.1. Mass Detector (MS)

A method was developed based on a capillary gas chromatography with mass spectrometry detection in selected ion monitoring (SIM) mode for the analysis of CIT, FLX, DCIT, DDCIT, and NFLX in urine samples. An optimized SPE procedure was applied which provided a preconcentration factor of 10, and no derivatization step was required [55].In order to achieve a high degree of separation for SER, PAR, CIT, DCIT, DDCIT, and DSER in a plasma sample, reextraction was done at varying pH, resulting in less interference from endogenous compounds. Such kinds of GC separations were performed using a cross-linked 30 m DB-35 ms silica capillary column, and helium (He) was used as a carrier gas (flow rate = 1.5 mL/min), with a mass spectroscopic measurement in ESI mode. The simultaneous chromatographic separation was achieved after the derivatization of analytes with reagent N-methyl-bis(trifluoroacetamide) [56]. SPME-GC has been used for the determination of SSRIs in urine samples. Fibers of 65 mm polydimethylsiloxane-divinylbenzene (PDMS-DVB) were used for the determination of the target SSRIs after simple in situ derivatization by acetylation of FLV, FLX, and SER. A 1.5 g aliquot of sodium chloride was then added to the samples. The vial was sealed with an aluminum cap and a teflon faced septum. It was then immersed in a water bath at 100°C and left to equilibrate for 5 min. To perform the extraction, the SPME fiber was exposed to the magnetically stirred sample for 30 min. The fiber was then immediately inserted into GC injection port and analysis was performed. Desorption time was set at 3 min. The time required for SPME step and for GC analysis (30 min each) enabled high throughputness [57].Derivatization is an important process which helps in large-scale screening of varieties of drug. This helps in the conversion of thermally labile and nonvolatile analytes to a suitable form for GC analysis, which has extended its use in the detection of antidepressant drugs and their metabolites. The separation of VLF, FLV, FLX, and CIT in blood samples was obtained by GC-MS operating in select ion monitoring (SIM) mode. Analytes were isolated by high yield LLE followed by derivatization throughtert-butyldimethylsilylation (MTBSTFA) and tri-methylsilylation (MSTFA) reagents. Thus, the analytes were analysed in tert-butyldimethylsilylated (TBDMS) and tri-methylsilylated forms (TMS) by GC-MS. Due to superior derivatization power and intensive derivatives, silylation was believed to have the best properties for large-scale screening with a variety of substances having structural differences. The method is feasible for highly sensitive, reliable, and routinely performed forensic toxicological analysis that produced satisfactory results with different concentration ranges of analytes in small sample volume [58]. In general, the derivatizing agents mostly preferred during GC separation of antidepressant drugs were N-methyl-N-trimethylsilyl-trifluoroacetamide for silylation, acetic anhydride-pyridine for acetylation, and trimethylammonium hydroxide for methylation [59].The identification and quantification of psychoactive drugs like antidepressants can be obtained using alternative biological matrices like oral fluid. Analyses of SER and PAR were obtained with GC-MS(-MS), operating in SIM mode after their SPE from oral fluid. Due to the basic nature of these drugs, eluting solvent used in SPE is a mixture of dichloromethane: isopropanol: ammonium (80 : 20 : 2, v/v/v). The chromatographic separation was performed on methylsilicone capillary column, and analytes were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and obtained retention time of 12.16 (SER) and 12.49 min (PAR). The method showed the applicability of alternative test matrices like oral fluid which have been extensively and successfully used to access acute and chronic exposure of drugs of abuse [60]. Similarly, in a suicidal case of multiple drug ingestion, CIT was identified and quantified in the postmortem samples by GC-MS. The concentrations of CIT found in femoral blood, heart blood, and urine were 0.88 mg/L, 1.16 mg/L, and 0.9 mg/L, respectively. This method also shows its applicability in the determination of CIT in postmortem tissue samples like liver, brain, gastric content, and so forth [61].Moreover, full-scan EI ionization mode is also considered as the reference technique for drug screening procedures since it plays an important role in the analysis of drugs in forensic toxicology and doping control. The main advantage of this technique is that it provides comparable GC-MS spectra on all commercially available instruments since the standardized ionization EI mode produces unique and uniform mass fragmentation patterns. As a consequence, large mass spectral libraries have been constructed which enable the identification of unknown compounds [62]. Table 2 represents the GC methods that appeared in the literature for the determination of new generation antidepressant drugs and their metabolites.Table 2
Gas chromatographic methods developed on for detection of SSRIs, SNRIs, and their metabolites.
Matrix
Analyte
Extraction       method
Detector system
Sensitivity
References
Mass detector
Urine
FLX, VLF, SER, and CIT
SPE
EI-MS
LOD, 2, 0.02, 1.25,  and 10μg/mL for FLX, VLF, SER, and CIT
[16]
Plasma
CIT, DCIT, DDCIT, FLX, and NFLX
SPE
MS
LOD = 0.7  to 33.6μg/mL
[55]
Plasma
CIT, DCIT, DDCIT, SER, DSER, and PAR
LLE
MS
LOQ = 2 ng/mL for CIT and PAR, 1 ng/mL for SER, 0.5 ng/mL for DSER, DCIT, and DDCIT
[56]
Urine
FLV, FLX, SER, VLF, and CIT
SPME
MS (ESI mode)
LOD < 0.4 ng/mL
[57]
Blood
VLF, FLX, FLV, CIT, and SER
LLE
MS (ESI in SIM mode)
LOD = <10, 25, 25, <10, 4 ng/mL, and LOQ = 25, 50, 25, 25, 50, 25, 20 ng/mL for VLF, FLV, LX, CIT, and SER, respectively
[58]
Oral fluid
SER
SPE
MS in SIM mode.
LOD = 18.6 ng/mL
[60]
Plasma
VLF, CIT, FLX, FLV, SER, PAR, DFLX, DSER, DCIT, and DDCIT
SPE
Mass (ESI and Chemical Ionisation  (PCI*/NCI* source) in SIM mode)
LOQ = 5 to 12.5 ng/mL in ESI and PICI mode and LOQ = 1 to 6.25 ng/mL in NCI mode
[97]
Plasma
CIT, DCIT, and DDCIT
LLE
MS
LOQ = 1 to 2 ng/mL
[98]
Other detectors
Serum
CIT and DCIT
LLE
NPD
LOQ = 10–20 mg/L
[63]
Blood
CIT
SPE
NPD
LOQ = 70 ng/mL
[99]
Urine
CIT, FLX, FLV, SER, and PAR
SPE
FID
LOQ = 91.7,  64.9, 100.3, and 61.4 and 95.1μg/L for CIT, FLX, FLV, SER, and PAR, respectively
[100]
Plasma
CIT, PAR, DLX, FLX, and SER
In-tube SPME
UV
LOQ = 50 to 500 ng/mL
[101]
Abbreviations*: PCI: Positive Chemical Ionization, NCI: Negative Chemical Ionization.


## 2.2.2. Nitrogen Phosphorous Detector (NPD)

Despite the differences in chemical structures of FLX, SER, CIT, VLF, DCIT, DSER, and NFLX,  were successfully assayed in serum samples after LLE, followed by GC-NPD. It has been observed that for GC procedure, polarity of extracting solvent (proportion of isoamyl alcohol in the mixture with hexane) was one of the most important variables influencing extractions. An added factor, which was also considered during analysis, is the nitrogen containing structures of analytes and responsible for the high sensitivity obtained using nitrogen phosphorous detector (NPD) (helps in minimisation of interferences). The method was reported with good linearity (20 to 400 mg/L) and correlation coefficients (r=0.998). The specificity of the method can help during the toxicological screening of antidepressant drugs [63]. Even stereoselective determination of FLX and NFLX enantiomers were obtained by using the two-dimensional capillary gas liquid chromatography with NPD that allows the separation of racemic mixtures of the drugs in plasma or serum. A three-step LLE was used for sample preparation and provided linear calibration between 5–250 ng/mL for (R)- and (S)-FLX and 15–250 ng/mL for (R)- and (S)-NFLX, with LOD = 1.5 ng/mL and 6 ng/mL, respectively [64].A comparative study was done for two types of extraction procedures regarding their recovery, precision, sensitivity, matrix purification efficiency, and less solvent consumption for the detection of SER and VLF in blood samples using GC-NPD. First procedure consists of the employment of chem elut column (diatomaceous earth) based on the principle of solid-liquid absorption extraction and closely related to conventional LLE, while in the second bond, elut certify column was used with mixed SPE having reversed-phase and cation-exchange sorbents. Recoveries of the compounds using chem elut columns at 500 ng/mL were in the range of 28–74% and with LOD and LOQ ranging from 39 to 153 ng/mL and from 128 to 504 ng/mL, respectively, while recoveries of the compounds using bond elut certify columns at 500 ng/mL were in the range of 64–86% and with LODs and LOQs ranging from 21 to 100 ng/mL and from 70 to 330 ng/mL, respectively. Thus, the results suggested that the use of reversed-phase and cation-exchange sorbent bond elut certify columns is more satisfactory for the screening of the selected drugs [65]. In a similar kind of study, the use of mixed-mode bonded-silica bond elut certify columns had shown advantages rather than chem elut columns, in terms of recoveries, cleaner extracts, sensitivity, precision and solvent consumption. It has also been explained that the chem elut column has diatomaceous earth support which was a hydrophilic packing porous material acted as a support for the aqueous phase. Its high surface area ensured that the organic eluents remain immiscible with the aqueous matrix, eliminating emulsion problems and facilitating an efficient interaction between sample and organic solvents, while bond elut certify column was a mixed-mode bonded silica extraction support containing hydrophobic chains and strong cation-exchange moieties. This gave the ability to retain acidic, neutral, basic, and amphoteric drugs on one column which is quite advantageous in the field of forensic toxicology [66]. Table 2 represents the GC methods in combination with various detectors that appeared in the literature for the determination of SSRIs and SNRIs and their metabolites.


## 2.3.  Electrodriven Methods

These are efficient and reliable analytical methods applied for the determination of enantiomers. Enantioseparations by capillary electrophoresis (CE) are generally achieved by adding a chiral selector to the running buffer, which reduces the cost of analysis with respect to some techniques such as HPLC chiral column. Cyclodextrins (CDs) are among the most prevalent selectors used in chiral CE. CE is one of the advantageous techniques used for the determination of small molecules in biological fluids which provides higher separation efficiency and selectivity in low sampling and solvent consumption. However, when biological fluids are injected directly into the capillary of CE unit, problems such as clogging of the capillary by particulate matter, irreversible binding of proteins, and peak distortion can occur because of the high salt concentration. Therefore, proper sample preparation or extraction is a must before analysis. The separation of SER, FLX, and FLV was described by CE with fully integrated SPE procedure. The SPE module was placed directly at the inlet of the capillary, and a mixture of all the analytes is extracted from aqueous solution by applying a simple pressure rinse. Under pressure-driven conditions, efficient elution was observed by using 50 mM phosphate buffer (pH = 3.5) in acetonitrile (20/80, v/v). It was observed that neutral monolith material in SPE gave an unstable electroosmotic flow, resulting in poor separation and elution. On the other hand, the addition of ionisable monomer (3-sulfopropyl methacrylate) to the SPE module has given improved and stable electroosmotic flow. This demonstrates the potential of inline SPE-CE for sensitive analysis of these drugs [67].CE, combined with LPME (porous polypropylene hollow fibres), was evaluated for chiral determination of CIT in plasma. The CIT enantiomers (R- and S-CIT) were extracted from 0.5 mL of plasma in alkaline medium (0.25 mL of 2 M NaOH). The unionised analytes were extracted into di-n-hexyl ether, impregnated in the pores of the hollow fibre, and into an acidic solution inside the hollow fibre and obtained excellent recoveries for both enantiomers (80%). This resulted in a three-phase system where the extracts were aqueous and hence directly compatible with the CE system. LOQ and LOD were found as 11.2 ng/mL and 3 ng/mL for both enantiomers, respectively. It has also been observed that the technique LPME-CE was a promising combination for the analysis of racemic drugs present in low concentrations in biological matrices [68].Similar kind of extraction procedure was also adopted in combination with CE, for the determination of CIT and its main metabolite N-DCIT in human plasma. The samples were made strongly alkaline which support proper extraction of the basic drugs like CIT. The drug and its metabolite were extracted into hexyl ether, impregnated in the pores of polypropylene hollow fibre, and into 25μL of 20 mM phosphate buffer (pH 2.75) present inside the hollow fibre (acceptor phase). This acceptor phase reduces the possibility of the majority of endogenic substances to penetrate inside hexyl ether layer. The instrument was operated at 20 kV, generating a current level of approximately 85 mA. LOQ and LOD for citalopram and N-desmethylcitalopram were found to be 16.5 ng/mL and 18 ng/mL and 5 ng/mL and 5.5 ng/mL, respectively [69].Aqueous and nonaqueous CE-MS in ESI mode were investigated for the separation of VLF and its metabolites. The separation of analytes at basic pH was found effective in resolving the drugs, but created considerable peak tailing. To overcome electrostatic interactions between analytes and silanol groups, experiments were carried out at acidic pH which lead to fine separation. Resolution was achieved in less than 3.5 min by the use of fused-silica capillary and a methanol-acetonitrile mixture (20/80 v/v) containing 25 mM ammonium formate and 1 M formic acid. It was also observed that the selectivity, efficiency, and separation time were greatly affected by the organic solvent composition. Nonaqueous CE was found as an appropriate alternative to resolve VLF and its metabolites simultaneously, as the electric current generated in nonaqueous medium was very low and allowed baseline resolution of VLF and its metabolites in 0.7 min only. However, the electric field was further increased by reducing the capillary length [70].Micellar electrokinetic chromatography (MEKC) with DAD was employed for the determination of CIT, SER, PAR, VLF, MCP, FLX, and their metabolites in urine samples. Here, the separation occurs on the basis of hydrophobic interaction with surfactant micelles. Separation and determination were optimised on an uncoated fused-silica capillary. The migration buffer consisted of 20 mM sodium borate at pH 8.5 with 20 mM sodium dodecyl sulphate and 15% isopropanol, at an operating voltage of 25 kV and column temperature at 40°C. Injection in the capillary was performed in hydrodynamic mode (0.5 psi, 15 s) and obtained migration time less than 11 min. Samples (1 mL) were extracted with diethyl ether (5 mL) at pH 9.6 and reconstituted in diluted migration buffer. LOD and LOQ were in ranges between 10 and 20 ng/mL and between 20 and 30 ng/mL, respectively, for all the molecules. This method allowed the determination of some of these analytes in biological fluids (blood and urine) obtained from postmortem cases that suggest the applicability of MEKC in forensic toxicological testing [71].Recently, citalopram and its main active metabolite were analysed in human urine samples through a new MEKC method. The samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C18 cartridge and by eluting the compounds with methanol. Best results were obtained by using 15 mM borate buffer (pH 9.2), containing 20 mM sodium dodecyl sulphate and 12% (v/v) 2-propanol as the background electrolyte. The separation was performed through a fused silica capillary with the application of hydrodynamic injection (pressure = 3.45 kPa) and 30 kV of separation voltage. Under these conditions, the migration times were observed in the range of 3.0 to 8.0 min, and the LODs were observed between 12.5 and 25 ng/mL, respectively [72].Since these compounds were produced in ionic form they can also be separated from capillary isotachophoresis (CITP). This is a technique used to separate charged particles, by using a discontinuous electrical field to create sharp boundaries between the sample constituents, thus creating separation. Conductivity detection of SER, CIT, FLX, and FLV has been done using CITP technique. This shows that it is a simple and quick method for the determination of these drugs [73]. An online preconcentration method, cation-selective exhaustive injection, and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC), with DAD, have been developed for the analysis of SSRIs. Under the optimized separation parameters, the enrichment factors for these five compounds when using CSEI-sweeping-MEKC fell within the range from 5.7 × 104 to 1.2 × 105, achieved LODs for the five SSRIs in human plasma between from 0.35 to 1.5 ng/mL [74].


## 3. Conclusion

The general trend observed in recent years for analysis of new generation antidepressant drugs is mostly emphasising separation techniques like liquid and gas chromatography in combination with various detectors. In this, the utmost analysis of has been observed with liquid chromatography in combination with mass, UV and fluorescence detectors. Gas chromatography has also been successfully applied with mass and nitrogen phosphorous detectors but required derivatisation of the concern drugs before analysis. This undeniably permits simultaneous analysis with high sensitivity and specificity and seemed to be suitable for their application in clinical and forensic studies. Moreover, in recent years, electrodriven techniques have been also becoming a viable alternative mean for analysis, providing high efficiency and short analysis time. In addition, for sample preparation, solventless microextraction techniques are evolved with tremendous possibilities and offer high sensitivity, accuracy, and enough reproducibility. However, despite of these advancements, efforts have still remained to focus on some alternative means of analysis which allow easier comparison and improved interpretation of results. Other chromatographic techniques like super critical fluid chromatography and ultra performance liquid chromatography would have become new analytical strategies especially for clinical and toxicological examinations and investigations of new generation antidepressant drugs.



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*Source: 139459-2013-10-31.xml*