A simple and reliable method for simultaneous quantification of antiepileptic drugsin plasma using LC-MS/MS combined with direct injection column# 1442

Kwang Youl Kim1, Yong Hyun Song1, Ju Hee Kang1,2, Cheol Woo Kim1,3, Moon Suk Nam1,41 Inha University Hospital Clinical Trial Center, 2Dep. of Pharmacology, Inha University School of Medicine, 3Dep. of Allergy and Clinical Immunology, 4Dep. of Internal Medicine, Inha University Hospital, Incheon, South Korea
Figure 3. Blank plasma chromatograms.
No interference peaks or matrix effects were observed in human plasmaABSTRACTRESULTS

A simple and robust method is presented for the simultaneous quantification of older and newer antiepileptic drugs (AEDs), including
lamotrigine, oxcarbazepine, zonisamide, gabapentin, topiramate, levetiracetam, phenytoin, carbamazepine, primidone in plasma by LCMS/MS. The method involved a rapid sample preparation by dilution with aqueous buffer followed by direct injection into a LC-MS/MSsystem. The separation is achieved on an Imtakt Cadenza HS-C18 HPLC column using gradient elution with a mixture of methanol andammonium acetate buffer.

Figure 1. Almost all of the plasma protein was already excluded from the column in this conditions.
HS-C18 can be used for the direct injection of samples containing body fluids.

Gabapentin

Oxcarbazepine

Primidone

Zonisamide

Levetiracetam

Carbamazepine

Topiramate

Phenytoin

Lamotrigine

Excluded
210 nm254 nmGood linearity (r=0.99, "1/x" weighting) was obtained for all drugs quantified over the range of clinically relevant concentrations in humanplasma and the use with two internal standards of sulfamethoxazole (positive ion) and tolbutamide (negative ion) improves accuracy andprecision for the analytical procedure. No interference peaks or matrix effects were observed in human plasma. The ruggedness of thismethod was examined about the reproducibility of column pressure, resolution, retention time and peak area with the greater than 500
injections of diluted plasma sample on a single column.

Cadenza HS-C18, 75 × 2.0 mm
A : 2mM ammonium acetate
B : MeOH
0% B (0-2min) ,0.35mL/min

This method is an excellent approach for the rapid preparation and high throughput of plasma samples containing AEDs drug levels in
the ug/ml range or lower, and therefore is the suitable method for daily therapeutic drug monitoring and pharmacokinetic studies ofAEDs

INTRODUCTION

Figure 4. Representative Calibration curves of AEDs

Figure 5. LC-MS/MS chromatogram of TIC (A) show that almost similar peak shape and resolution of all
AEDs at injection times above 500 and also pressure change (B) of HS-C18 column is constant.

Table 2. Precision and Accuracy of Inter-day (n=3)

BA
Table 1. MRM transitions and Optimized parameters

A simple and reliable method for simultaneous quantification of antiepileptic drugs (AEDs) in human plasma sample is necessary for
efficient therapeutic drug monitoring of patients treated with AEDs polytheraphy. LC-MS/MS assay offers selective and sensitivetechniques to quantify the drugs of interest in complex biological samples such as plasma. However, plasma samples still need intensive
sample handling and clean-up prior to LC-MS/MS analysis.

RSD = 4.8%

Dilute-and-shoot preparation is commonly used for the determination of drugs from biological matrices such as urine, but is not typicallyused with plasma samples because the amount of protein present in plasma can cause a variety of problems including analytical column
failure and matrix effect (mostly ion suppression). In pharmaceutical analysis, pretreatment steps such as automated on-line or off-line
solid phase extraction and column switching are often used to remove or minimize matrix effect, but these method development is timeconsuming and expensive.
The new direct injection column (Cadenza HS-C18) is containing a silica based hybrid ODS stationary phase with both C18 andhydrophilic groups which is responsible for excluding proteins in biological matrices. It can exclude proteins from biological matrices andalso retain drugs by running a linear gradient of the organic solvent with 100% aqueous buffer as the initial solvent. Therefore, it can beused for the direct injection of samples containing body fluids such as plasma using divert valve.
In this work, we developed an LC-MS/MS method combined with direct injection column to quantify nine AEDs in human plasma withconstant polarity switching in a single analytical run.

500th
200th
100th

Table 3. Precision and Accuracy of Intra-day (n=3)

CONCLUSIONS

Figure 2. TIC chromatogram (The divert valve was switched to the MS at 1 min ~3 min & 4.5 min ~ 6.5min
and to waste other time) and MRM chromatograms of all compounds

1. An efficient, simple and accurate LC-MS /MS method was developed for the quantification of nine AEDs in human plasma to support
daily TDM and pharmacokinetic studies.

MSMS

Sulfamethoxazole

MATERIALS AND METHODS
Sample preparation
10 µL of plasma was diluted with 500 µL 2mM ammonium acetate buffer containing internal standards in concentration of each 1ug/mL
for sulfamethoxazole and tolbutamide. Sample was vortexed for 15 sec and centrifuged for 5 min at 13000 rpm and finally 1 µL ofsupernatant was injected onto LC-MS/MS system.
Liquid Chromatography
LC separation of all analytes was achieved on a Imtakt Cadenza HS-C18 column (3um, 2.0 × 75 mm) at 30 °C using a Agilent 1200 LCsystem. A gradient condition was used with mobile phase A (2mM ammonium acetate buffer) and mobile phase B (Methanol) as follows :The all analytes were eluted at a flow rate of 350 μL/min.
Mass Spectrometry
Mass spectrometric detection was peformed with an API 4000
triplequadrupole mass spectrometer (MDS SCIEX, Toronto,Canada) operated in multiple reaction monitoring (MRM) mode
constant (700ms) positive/negative polarity switching. Ionization of
all analytes was carried out using the electrospray ionization (ESI)and TurboIon Spray (TIS) source temperature was maintained at
350 °C. Both the nebulizer (GS1) and TIS (GS2) were set at 50 psi.
Mass parameters about analytes were optimized as as showntable 1. Data processing was performed on Analyst 1.5.1 software(SCIEX).

2. The ruggedness of the dilute-and-shoot method was evaluated with greater than 500 injections on a single column, this method is anexcellent approach for the rapid preparation and high throughput of plasma samples containing drug levels in the ng/ml range or higher.

Table 4. Validation data of calibration samples
Gabapentin

Primidone

Table 5. Matrix effect (any change in theionization process of the AEDs)

3. HS-C18 column provides excellent separation for low molecular weight analytes especially hydrophobic compounds in protein samples.

REFERENCES
Levetiracetam

1. Laura Mercolinia, Roberto Mandriolia, Mario Amoreb, Maria Augusta Raggia,(2010) Simultaneous HPLC-F analysis of three recent
antiepileptic drugs in human plasma .Journal of Pharmaceutical and Biomedical Analysis 53 (2010) 62–67

Topiramate

2. Levy, R.H; Mattson H.; Meldrum, B.S.; Perucca, E.: Antiepileptic drugs, 5th edition, Lippincott Williams & Wilkins
Tolbutamide

Zonisamise

Oxcarbazepine

Phenytoin

Carbamazepine

Lamotriginea ME (%)=[(Area spiked sample / Area std solution) -1] × 100 (%)