Derayea et al. BMC Chemistry(2024) 18:137
https://doi.org/10.1186/s13065-024-01240-0

BMC Chemistry

RESEARCH

Open Access

Development and validation of a precise
flow injection method for the assessmentof brexpiprazole, with application
to pharmaceutical dosage forms and humanplasma analysis
Sayed M. Derayea1, Al Amir S. Zaafan2*, Dalia M. Nagy1 and Mohamed Oraby2
Abstract
A novel antipsychotic medication named brexpiprazole (BRX) is currently employed for the treatment ofschizophrenia and other psychotic disorders. Because BRX’s molecular structure includes a benzothiophene ring,
it natively fluoresces. To detect BRX with precision and speed, a flow injection-fluorometric method, which isboth sensitive and selective, is recommended. The fluorescence detection was conducted at 364 nm following
excitation at 326 nm to capture the strong intrinsic fluorescence of BRX. The carrier solution employed was a
mixture of phosphate buffer (pH 4, 10 mM) and acetonitrile (50: 50, v/v), with a flow rate of 0.5 mL min− 1. The
calibration curve, based on peak areas, exhibited linearity within the concentration range of 20–350 ng mL− 1,with a remarkable correlation coefficient (r2) of 0.9999. The limit of quantitation was 9.7 ng mL− 1, and the limit
of detection was found to be 3.2 ng mL− 1. This method was applied to quantify BRX in Neopression® tablets,
achieving recovery within an acceptable range without interference from the tablet’s additives. Additionally,
the proposed approach was successfully utilised to quantify the drug in spiked human plasma. The approach
underwent validation following ICH requirements.
Keywords Brexpiprazole, Flow injection analysis, Fluorescence, Pharmaceuticals, Human plasma*Correspondence:
Al Amir S. Zaafan
alamirsaber428@gmail.com
1
Analytical Chemistry Department, Faculty of Pharmacy, Minia University,
Minia 61519, Egypt
2
Department of Pharmaceutical Analytical Chemistry, Faculty of
Pharmacy, Sohag University, Sohag 82524, Egypt© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Derayea et al. BMC Chemistry(2024) 18:137

Introduction
Brexpiprazole (BRX), depicted in Fig. 1, is a 7-4-[4-(1benzothiophene-4-yl) piperazin-1-yl] butoxy-1, 2- dihydroquinoline-2-one. BRX is categorized as an atypicalantipsychotic medication. It has been granted approval
as a therapeutic agent for schizophrenia and as a complementary therapy for major depressive disorder [1].
The FDA granted its therapeutic use for schizophrenia and depressive disorders in July 2015. BRX exhibits
both blocking and activating effects at its target receptor,
offering potential benefits in reducing restlessness andagitation due to its predominant blocking and diminished stimulating properties [2]. It also acts by modulating the activity of serotonin and dopamine. Functionally,
BRX is considered as a new drug that stimulates partially
1 A serotonin and D2 dopamine receptors, it is also a
powerful blocker of serotonin 2 A receptors, and anantagonist of noradrenergic alpha 1B and 2 C receptors[3]. BRX’s higher effectiveness allows for the use of lower
doses compared to other antipsychotic drugs. Notably, its
affinity for serotonin 5HT1A receptors is notably higher,
leading to fewer adverse effects, such as akathisia andextrapyramidal symptoms, when compared to other antipsychotic medication classes [4].
Existing analytical methods for BRX, as revealed in theliterature review, encompass spectrophotometric [5–7],
spectrofluorimetric [8], HPLC [2, 3, 7, 9–11], TLC [2,12], and electrochemical method [13]. These approaches
require thorough sample preparation, substantial quantities of pure organic solvents, and lead to increased
analytical expenses and environmental concerns. Spectrophotometric techniques, though less sensitive, areseldom used in the examination of biological fluids [14].
In contrast, chromatographic methods, while extremely
sensitive and suitable for the study of drug degradation products and pharmacokinetics, are hindered bytheir time-consuming nature, the need for expensive,advanced equipment, significant quantities of exceptionally pure organic solvents, and the requirement for welltrained personnel [15–17].
Consequently, this study aims to introduce a novel
approach by developing a flow injection method forFig. 1 The chemical structure of BRX

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BRX quantification, marking the first instance of sucha method for BRX measurement. This approach offersa time-saving, sensitive, and solvent-efficient alternative for evaluating BRX in tablet form and spiked humanplasma. The procedure validation adheres to the (ICH)
recommendations [18]. The methods that using spectrofluorimetric detectors are highly selective due to thepresence of two spectra, extremely sensitive, and needvery easy sample preparation [19–21]. Flow analysis has
become a crucial field in modern analytical chemistry. Its
current importance is due to the comprehensive development of specialized instrumentation, its many advantages
compared to conventional methods, a wealth of scientificand patent documents, and the abundance of applications tailored to diverse fields such as chemical analysis,
food and agricultural chemistry, as well as environmental
and clinical chemistry [22].

Experimental
Instrumentation

The flow injection setup utilized in this study compriseda Sykam S 1130 HPLC quaternary pump equipped with
an optional built-in vacuum degasser (Sykam GmbH,
Gewerbering, Germany). Samples were introduced into
the system using a Hamilton HPLC syringe (Franklin,
MA, USA). To carry out fluorometric measurements,
a detector RF-20 A (Shimadzu, Kyoto, Japan) was connected to the HPLC system. It’s important to note that, in
this study, the HPLC system’s column was not employed.
Aquatron water still A4000D was used to produce double-distilled water (Cole-Parmer, Staffordshire, UK). The
pH was regulated with a Jenway 3510 pH meter (Staffordshire, UK). A Mettler Toledo 5-digit balance (Greifensee,
Switzerland) was utilized for weighing.
Materials and chemicals

The BRX pure powder was received as a generous donation from Marcyrl Pharmaceutical Industries (Obour
City, Cairo, Egypt). We procured acetonitrile and methanol of analytical quality from Sigma Aldrich (Darmstadt,
Germany). The sources for orthophosphoric acid anddipotassium hydrogen phosphate were El Nasr Chemicals

Derayea et al. BMC Chemistry(2024) 18:137

Chemical Co (Cairo, Egypt). Additionally, Neopression®
4 mg tablets (Batch no. 2,134,327) were obtained fromthe Egyptian marketplace. Lastly, Whatman grade No.
1 filter papers (Maidstone, Kent, United Kingdom) were
utilized.
Flow injection conditions

The method was carried out with a carrier solution that
was a combination of (50: 50, v/v) acetonitrile and phosphate buffer (pH 4, 10 mM). Prior to use, this mixture
was filtered through a 0.22 μm filtration membrane anddegassed. The liquid flowed through the system at a flowrate of 0.5 mL min− 1. The native fluorescence of BRX wasdetermined at an emission wavelength of 364 nm following excitation at 326 nm. Using an injection volumeof 20 µL, the whole procedure was performed at room
temperature.
Preparation of carrier solution

Approximately, 0.871 g of dipotassium hydrogen phosphate was transferred into a 500 mL volumetric flask.
Subsequently, 500 mL of double-distilled water wasadded in the flask, and the mixture was thoroughly
shaken. The pH of the resulting solution was modified toreach a value of 4.0 with orthophosphoric acid. Following
this, the solution underwent filtration through a 0.22 μm
filtration membrane, and any remaining air was removed
by subjecting the solution to 5 min of sonication. Finally,
it was blended with acetonitrile (50: 50, v/v).
Preparation of standard stock solution

A 1 mg mL− 1 stock solution was formed by precisely
measuring 10 mg of pure BRX and dissolving it in 10 mLof methanol. For the BRX working standard solution,
the carrier solution was used. The stock solutions werestored in the refrigerator. Subsequently, BRX solutions
with concentrations of 20, 50, 100, 150, 200, 250, 300, and
350 ng mL− 1 were generated through a series of dilutions
using the carrier solution.
Preparation of pharmaceutical tablets

Ten tablets of Neopression® 4 mg were finely ground. An
accurately measured amount of tablet powder, equivalent to 4 mg of BRX, was employed. The BRX was thenextracted using analytical-grade methanol in a volumetric flask through sonication lasting 30 min. Subsequently,
the volume was adjusted to 50 mL with the same solvent.
Whatman grade No. 1 filter paper was utilized to filter
the resultant solution, with the initial portion of the filtrate being discarded. For the preparation of the assessed
concentration, the extract was diluted using the carrier
solution. Five measurements of BRX concentration were
determined following the standard analytical procedure.

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The tablets’ actual content was determined using the corresponding regression equation.
Preparation of spiked human plasma

Blood samples were collected from the forearm veinsof healthy volunteers at Misr Hospital in Sohag, Egypt.
These samples were placed in tubes containing heparin.
Prior to experimenting, the purpose of the study was
explained to each volunteer, and they provided their written consent. The use of plasma specimens from healthy
individuals was in accordance with the Helsinki Recommendations [23, 24].
To conduct the experiment, 100 µL of drug-free humanplasma was mixed with 100 µL of various concentrations of BRX. This mixture was then treated with 200 µlof acetonitrile to precipitate proteins. The solution wascentrifuged for 30 min at 4000 rpm following a powerful 30-second vortex. Clear supernatant samples weretaken, and a standard analytical procedure was applied.
This methodology was employed to assess the drug underinvestigation at four different concentrations, with three
replicates for each concentration.

Result and discussion
Detection wavelength selection

To select an appropriate analytical wavelength, a solution
containing 10 µg mL− 1 of BRX was prepared by diluting
a standard solution. Specifically, 10 mg of standard BRX
powder was placed in a 10 mL volumetric flask and dissolved in a small volume of methanol. The flask was thenfilled up to the mark using the same solvent. From thissolution, 0.1 mL was withdrawn and transferred into
a separate 10 mL volumetric flask, again brought up to
the mark with the same solvent, resulting in a concentration of 10 µg mL− 1. This solution was analyzed using adouble-beam spectrophotometer in the range of 200 to
400 nm, with methanol as the blank. The resulting spectrum revealed that the maximum absorption wavelength(λmax) of BRX was 326 nm, which was chosen as the excitation wavelength for the subsequent analysis. To prepare
a lower concentration standard solution of 0.5 µg mL− 1
of BRX, methanol was also used to make dilution of thestandard solution appropriately. This diluted solution wasthen examined for its emission wavelength using a spectrofluorometer after excitation at 326 nm. It was determined that BRX exhibited its maximum fluorescence at
364 nm, and this wavelength was selected for the emission analysis.
Carrier solution selection

The BRX standard solution was introduced into theequipment and tested with various solvent systems. To
determine the best solvent system for BRX, we explored
several options, including methanol, acetonitrile, andDerayea et al. BMC Chemistry(2024) 18:137

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Fig. 2 BRX FIAgram 50, 150, 250, 300, and 350 ng mL-1

Table 1 Regression equation and validation parameters for the proposed flow injection method
Parameters
Linear range (ng mL− 1)
Slope (b)Regression Equation
Standard deviation of slope (Sb)Intercept (a)Standard deviation of intercept (Sa)
Correlation coefficient (r)Determination coefficient (r2)
LOD (ng mL− 1)
LOQ (ng mL− 1)

water in different combinations and pH conditions. The
most intense fluorescence occurred when we combineda buffer with acetonitrile. To identify the ideal proportion of these two components for the method, we experimented with various ratios, such as 20:80, 40:60, 50:50,
70:30, 80:20, and 90:10. We discovered that the mosteffective carrier solution system is a 10 mM phosphatebuffer and acetonitrile in a 50:50 volume-to-volume ratio.
We also tested different pH levels and found that pH 4
yielded the highest fluorescence (Fig. 2).
Optimization of the flow rate

The FIA system’s flow rate plays a crucial role as it has anotable impact on the peak area, which reflects the fluorescence intensity response. To optimize the flow rate,various rates (0.5, 0.8, 1, and 1.2 mL min− 1) were tested.
Interestingly, it was noticed that increasing the flow rate
led to a substantial decrease in the peak area. Consequently, 0.5 mL min− 1 was determined to be the optimal
flow rate, as it yielded the highest peak area with a sharp
peak.

BRX
20–350141.21
Y = 141.21X + 2767.73
0.66
2767.73137.550.99990.99983.21
9.74

Validation of the suggested method

Based on ICH Q2 (R1) guidelines, the validated analytical procedure [18] was assessed in terms of parameterssuch as linearity, detection limit (LOD), quantitation
limit (LOQ), precision, accuracy, robustness, and similar
attributes.
Linearity

Several typical solutions of the studied medication wereanalyzed at different concentrations using the standard
analytical procedure. By plotting the peak area against
the eventual drug concentration, the calibration curve
was established for the drug. The linearity plot for the
proposed assay of BRX covered a concentration rangeof 20–350 ng mL− 1, displaying an impressive correlation
coefficient (r2) of 0.9999. The regression data, calibration data, and correlation coefficients are summarized inTable 1.

Derayea et al. BMC Chemistry(2024) 18:137

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Table 2 Precision for proposed flow injection method for the determination of BRX
Precision level

Added concentration (ng mL-1)

Measured concentration (ng mL-1)

Intra-day(Day1, n = 3)

100
200
300
100
200
300

100.69196.70297.27100.02
199.26300.60Inter-day(three days, n = 9)

Recovery*(%)
100.6998.35
99.09
100.02
99.63
100.20Standard deviation

RSD (%)

1.06
0.14
0.48
1.33
1.26
0.93

1.05
0.14
0.49
1.33
1.26
0.93

Standard deviation
0.92
1.11
1.08
0.67
0.32

RSD (%)
0.92
1.10
1.09
0.66
0.32

*Mean of three determinations
RSD Relative standard deviation

Table 3 Evaluation of the accuracy of the analytical procedure by standard addition method (n = 3)
Amount taken (ng mL− 1)
100
100
100
100
100

Amount added (ng mL− 1)
0.00
50
100
150
200

Amount found (ng mL− 1)
99.80
151.47198.40252.60301.02

Recovery* (%)
99.80
100.9899.20
101.04100.34*Mean of three determinations
RSD Relative standard deviation

Limit of detection and limit of quantitation

The LOD represents the minimum detectable amountwithin a sample under the specified experimental conditions, although it is not measurable with precision. On
the other hand, the LOQ signifies the smallest amount of
BRX in a sample which can be measured with a satisfactory level of precision and accuracy. To determine thesevalues, the following formulas were employed: LOD = 3.3
times the standard deviation of the intercept (SD) divided
by the slope of the calibration curve (S). LOQ = 10 (SD)/S. The values for LOD and LOQ can be found in Table 1.
Precision

The study examined the precision of BRX, both within
the same day (intra-day) and across different days (interday), using three concentrations: 100, 200, and 300 ng
mL− 1. To assess intra-day precision, three consecutive
analyses were performed on the same day, whereas interday precision was evaluated by performing the analyticalprocedure over three successive days. This experiment
was conducted three times. The recovery values obtained
ranged from 98 to 102%, and (% RSD) values wereobserved to be well within acceptable limits, not exceeding 2.0%. A summary of the results for both intra- andinter-day precisions can be found in Table 2.
Accuracy

The BRX recovery was determined by employing thestandard addition technique to evaluate the method’s
precision. Various quantities of the standard solution were introduced into tablet solutions previously
analyzed. This recovery assessment encompassed five
distinct concentration levels and was conducted intriplicate. The results, including the calculated percentage recoveries and associated SD, are presented inTable 3. The average BRX recoveries ranged from 98 to
102%, and the standard deviations remained below 2.0%.
These findings demonstrate that the proposed methodology is sufficiently accurate.
Robustness

The method’s robustness was evaluated by examining its
performance when subjected to minor adjustments in theexperimental parameters. These included slight variations in the carrier solution composition (2%), minor
fluctuations in flow rate (0.1 mL min− 1), changes in pH(0.1), and shifts in the detection wavelength (2 nm). The
findings are summarized in Table 4. Since the obtained
RSD remained below 2%, it is evident that the method
remained largely unaffected by these minor variations in
the tested variables. This confirms the robustness of the
proposed technique.
Applications of the method
Application to pharmaceutical dosage forms

An analysis of the drug content in a commercially available dosage form, Neopression® 4 mg tablets, was conducted using a general analytical method, illustrated
in Fig. 3. The proposed approach yielded a satisfactory
percentage recovery of 98.73 ± 1.42. This notably highpercentage recovery also indicated that the tablet’s nonactive ingredients did not have a discernible impact. Furthermore, the results obtained through the suggested
approach were compared to those from a previously
published method [7] in terms of precision and accuracy.
The calculated t- and F- tests values, at a 95% confidenceDerayea et al. BMC Chemistry(2024) 18:137

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Table 4 Robustness for 200 ng mL-1 of BRX (n = 3)
Parameter
Flow rate
Percentage of acetonitrile (%)
pH
λ excitation (nm)
λ emission (nm)

0.4
0.6
48
52
3.9
4.1
324
328
362
366

Recovery͙͙* (%)
101.51
98.79
99.89
100.24101.29100.27
98.52
99.11
98.66
100.22Standard deviation
1.67
0.62
0.40
1.11
0.80
0.87
0.16
0.79
0.30
0.52

RSD (%)
1.65
0.63
0.40
1.11
0.79
0.87
0.17
0.80
0.31
0.52

*Mean of three determinations
RSD Relative standard deviation

Fig. 3 BRX (150 ng mL-1) FIAgram in dosage form

Table 5 Analysis of BRX in dosage form (Neopression 4 mg/ tablet) and pure form by reported and proposed methods [7]Parameters% Recovery aStandard deviation
Variance
Number of measurements
t-test b
F-test ba

Dosage form
Proposed method
98.73
1.419
2.014
5
1.11
1.32

Reported method
100.01
1.624
2.637
5
---

Pure form
Proposed method
99.46
1.23
1.51
5
0.75
1.26

Reported method
98.84
1.38
1.90
5
---

Mean of five determinationsb

Tabulated value at 95% confidence limit, F = 6.388 and t = 2.306

level, did not exceed the corresponding tabulated values,
as demonstrated in Table 5. Hence, no significant disparity was observed between the outcomes of the two
methods.

Application to spiked human plasma

The highly sensitive FIA method effectively detected
BRX in human plasma samples spiked with varying
medication concentrations. To achieve this, different
drug concentrations were mixed with human plasma,
and acetonitrile was introduced to precipitate proteins.

Derayea et al. BMC Chemistry(2024) 18:137

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Fig. 4 BRX FIAgram in human plasma 50, 100, 200, 300 ng mL-1

Table 6 The application of the developed method to measure the drug in spiked human plasma
Drug conc. (ng mL− 1)
50
100
200
300

% Recovery*
102.53100.3399.17
100.98

SD
2.26
1.11
1.28
0.48

%RSD
2.20
1.11
1.29
0.47

*Mean of three determinations, SD standard deviation, RSD Relative standard deviation

Following centrifugation, the recommended analytical
protocol was applied. BRX exhibited excellent recovery rates (Fig. 4), and the impact of interfering plasmacomponents was notably reduced, as illustrated inTable 6. The respective drug concentrations were determined by utilizing the resulting regression equation(Y = 141.21X + 2767.73).
Comparison with the reported methods

A comparison of the analytical performance of the suggested FIA method with other reported methods,
including chromatography and spectrometry (Table S1),
demonstrates that, the current method has a very low
limit of detection (3.21 ng mL− 1). Although a few of thepreviously reported methods had higher sensitivity, thesensitivity obtained from the current work is satisfactory
for the quantification of BRX in biological samples andinvestigating the pharmacokinetic behavior of BRX. The
greenness of the proposed method is not very high due
to usage of acetonitrile solvent, however, it has higher
greenness than reported chromatographic methods thathave higher sensitivity [7, 9]. Meanwhile, the current
method is characterized by ease of analysis, highly simple and selective procedure. Furthermore, no extensive

sample treatment is needed and is cost-effective. All
these merits make the present method highly convenient
for routine analysis in quality control laboratories.

Conclusion
This study introduced a new flow injection-fluorometrictechnique which was utilized to measure BRX in bulk
materials, pharmaceutical dosage forms, and humanplasma samples that were intentionally spiked with thecompound. The suggested approach has been highlighted
by its ease of use, sensitivity, precision, and accuracy. It
demonstrated selectivity and reproducibility in the analysis of BRX, showing no interference from additives asconfirmed through statistical analysis. The procedure
effectively quantified BRX in pharmaceutical dosage
forms, producing results closely matching the labeledvalues of commercially available tablets. Moreover, its
high sensitivity makes it suitable for analyzing biologicalsamples and quantifying BRX in spiked human plasma.
The method’s validity was confirmed by ICH guidelines.
Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01240-0.

Derayea et al. BMC Chemistry(2024) 18:137

Page 8 of 8

Supplementary Material 1
8.
Acknowledgements
Not applicable to this study.
Author contributions
Sayed M. Derayea: Conceptualization, supervision, reviewing, writing, andediting. Al Amir S. Zaafan: Conceptualization, methodology, software, andpreparation of original draft writing. Dalia M. Nagy: data curation, visualization,and Mohamed Oraby: Methodology, validation, reviewing, and writing.

9.

Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).

11.

Data availability
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

13.

Declarations

14.

Ethics approval and consent to participate
All experimental protocols in the current study were approved by theCommittee of Research Ethics in the Faculty of Pharmacy, Sohag University,
Sohag, Egypt. All methods were carried out in accordance with relevant
regulations and guidelines. In all cases, informed written consent was
obtained from each participant before donating the blood samples.
Consent for publication
Not applicable.

10.

12.

15.

16.
17.

Competing interests
The authors declare no competing interests.
Received: 14 March 2024 / Accepted: 4 July 2024

18.
19.

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