Quantitative methods in the analysis of clozapine in human matrices – A scoping review

Jia Le Lim; Mogana Rajagopal; Gabriel Akyirem Akowuah; Fazlollah Keshavarzi; Khaled Mohammed Ahmed Alakhali

doi:10.1515/revac-2023-0066

Article Open Access

Quantitative methods in the analysis of clozapine in human matrices – A scoping review

Jia Le Lim , Mogana Rajagopal , Gabriel Akyirem Akowuah , Fazlollah Keshavarzi and Khaled Mohammed Ahmed Alakhali

Published/Copyright: March 21, 2024

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From the journal Reviews in Analytical Chemistry Volume 43 Issue 1

Abstract

Clozapine (CLZ) has retained its clinical utility in the management of schizophrenia despite the discovery of novel antipsychotics, as it possesses unique efficacy in the setting of treatment resistant schizophrenia while causing minimal extrapyramidal symptoms. However, these benefits are offset by the risk of agranulocytosis and other side effects, and therapeutic drug monitoring (TDM) is routinely recommended for patients undergoing treatment with CLZ. A multitude of approaches for the quantification of CLZ have been developed for different settings such as TDM, quality control of pharmaceutical dosage forms, and toxicology studies. Primarily, these approaches fall under one of three branches of analysis, namely, chromatography, electrochemical analysis, and spectrophotometry. This study provides a scoping review of the recent advances in the methods of quantification for CLZ and highlights the potential utility of novel methods in the field of drug quantification.

Keywords: clozapine; scoping review; analysis; quantification; chromatography

1 Introduction

Clozapine (CLZ) is an atypical antipsychotic used primarily in the treatment of schizophrenia. Despite the discovery of a number of novel antipsychotics in recent years, CLZ still holds a significant clinical importance due to its effectiveness in managing treatment-resistant schizophrenia (TRS), the positive and negative symptoms of schizophrenia, as well as preventing suicidal tendencies in schizophrenic patients [1]. Unfortunately, the benefits of CLZ are equally offset by its risk of rare but severe side effects that are not present in other antipsychotic medications. Primarily, patients are prone to severe neutropenia which can occur in early stages of CLZ therapy. Other side effects of CLZ include myocarditis, cardiomyopathy, sialorrhea, and tachycardia. A slow dosage titration is also required as large or sudden increases in CLZ dose have led to cardiovascular collapse and death, especially in patients taking benzodiazepines concomitantly [2]. Thus, a consensus guideline for psychotropic drugs published in 2017 strongly recommends therapeutic drug monitoring (TDM) for all patients treated with CLZ, with the aim to achieve a therapeutic range of 350–600 ng·mL⁻¹ [3]. Historically, TDM of clozapine has been carried out with high-performance liquid chromatography (HPLC) assays due to its favourable accuracy, precision, and ease of use. However, given that TDM plays a significant role in CLZ therapy, novel techniques of analysis such as mass spectrometry (MS), ultrahigh-performance liquid chromatography (UHPLC), and novel radioimmunoassay have been developed in recent years which may improve the efficiency and utility of TDM in clinical practice. TDM is also largely influenced by a drug’s pharmacokinetics. Although it has been several decades since its discovery, recent studies have unveiled new information on CLZ pharmacokinetics, which provides insight to improve its clinical utility. The aim of this study is to provide a scoping review which explores the methods available for quantification of CLZ in various human matrices.

2 Methods

The scoping review was carried out based on a systematic approach to identify and summarize studies related to the methods of quantification of CLZ. As outlined by the framework proposed by Arksey and O’Malley [4], this study is carried out in five stages which includes identifying the research question, identifying relevant studies, study selection, charting data, followed by interpreting and reporting the results. The research question of this study was: “what are the methods of analysis that have been developed for the quantification of CLZ in human matrices?” Following that, we identified the keywords from the research question, namely, CLZ, quantification, human matrices. Preliminary literature search also showed that the most common methods of quantification fall into either chromatographic, electrochemical, or spectrophotometric techniques. Therefore, we decided to include the keywords chromatography, electrochemical, and spectrophotometry into the search terms. To obtain a comprehensive set of results with the resources available, a strategy was developed to identify relevant studies based on a set of inclusion and exclusion criteria, as shown in Table 1. The search was carried out in PubMed and Google Scholar using the search term: “(clozapine) AND (quantification OR analysis) AND ((human matri*) OR (urine) OR (plasma) OR (nail) OR (blood)) AND ((chromatograph* OR electrochemical OR spectrophotometr*) OR ((HPLC) OR (UHPLC) OR (GC) OR (LC-MS) OR (MS))) AND (1993:2023[pdat]).” The selection process of papers is illustrated in Figure 1. The initial search yielded a total of 5,160 results (4,860 from Google Scholar and 300 from PubMed). Duplicates were removed from the results to yield 4,756 papers, of which 92% were excluded based on screening of title and abstract. Following that, 375 papers were screened according to the inclusion and exclusion criteria, and a total of 45 papers were included in this study.

Table 1

Inclusion and exclusion criteria employed in the literature search

	Inclusion criteria	Exclusion criteria
Time period	Articles published between 1993 and 2023	Articles published before 1993
Language	English articles	Non-English articles
Focus of the paper	Quantification of CLZ in human matrices	Topics that are not relevant to the quantification of CLZ in human matrices
Research type	Primary research	Secondary research, including review articles, scoping reviews, and systematic reviews
Publication type	Articles published in peer reviewed journals	Articles published in non-peer reviewed journals, websites, newsletters

Figure 1

Flow chart of the search strategy and results.

3 Analytical methods for CLZ

To date, a multitude of techniques have been developed for the quantification of drug compounds in human matrices. Chromatographic methods such as HPLC and gas chromatography (GC) make use of mobile phases and stationary phases with contrasting properties to separate and quantify chemical compounds [5,6,7,8,9,10,11]. Due to their high sensitivity and precision, in addition to being relatively easy to carry out, chromatographic methods of quantification are widely used in the clinical setting [12,13,14]. Meanwhile, electrochemical methods of drug quantification rely on passing current through a set of electrodes, which allows the drug compounds to accumulate and subsequently quantified [15,16]. Spectrophotometry is another widely studied and utilized method for drug analysis, whereby specific chemicals are allowed to complex with the drug molecule, which can then be quantified using a spectrophotometer [17–19].

3.1 HPLC/UHPLC

HPLC systems operate on the separation of chemical entities based on their different physiochemical properties, such as polarity, particle size, and binding affinity [20–22]. In the analyses of CLZ, reverse-phase HPLC remains the hallmark mode of HPLC for quantification. CLZ is largely non-polar in nature and elutes relatively quickly in reverse-phase chromatography. This results in timely and efficient analyses to be carried out. UHPLC systems are optimized to operate at much higher column pressure than HPLC and provides much better separation and quicker analysis [23]. Despite the advances in other methods of drug analysis, HPLC is still widely used owing to its reliability, sensitivity, and relative ease of use. The summary of HPLC and UHPLC methods of CLZ quantification are shown in Table 2.

3.2 GC-MS

GC is an alternative chromatographic method which have been studied for the quantification of CLZ and various other drugs [10,24,25]. In an ideal system, GC systems provide good sensitivity and accuracy at a relatively low running cost. However, GC analyses are generally only applicable to volatile and non-heat sensitive analytes, which severely limits its utility and application to drug analysis [11,26]. GC methods can also be coupled with mass spectrometry to further analyse the results. GC separates the different components of a mixture, while MS further separates the components of each peak based on their mass-to-charge ratio. This coupling allows the identification of unknown analytes, determine structural and chemical properties, and study of the breakdown of the analytes [27–29]. Table 3 shows the overview of GC methods for CLZ quantification.

Table 2

Summary of HPLC and UHPLC methods of CLZ quantification

Stationary phase	Analyte	Matrices	Mobile phase	Detection (nm)	Retention (min)	Linearity (ng·mL⁻¹)	LOD (ng·mL⁻¹)	LOQ (ng·mL⁻¹)	Ref.
Spherisorb S5 SCX (sulphopropyl-bonded silica; phase separation) cation exchange column (150 × 4.6 mm ID)	CLZ and Norclozapine (NCLZ)	Plasma and serum	Methanol containing ammonium perchlorate, pH 6.7	215	CLZ: 5	500–1,500	CLZ: 50	—	[13]
	CLZ and Norclozapine (NCLZ)	Plasma and serum	Methanol containing ammonium perchlorate, pH 6.7	215	NCLZ: 12	500–1,500	NCLZ: 50	—	[13]
LiChroCART Superspher 60 RP-select B (250 cm × 4 mm ID, 4 µm)	Citalopram, CLZ, fluoxetine, norfluoxetine, maprotiline, desmethylmaprotiline and trazodone	Serum	Buffer/CH₃CN (70/30, v/v)	260	CLZ: 2.5	—	CLZ: 10	CLZ: 29.9	[49]
C18 STR ODS-II column, (150 mm × 4.6 mm ID, particle size 5 m)	CLZ and NCLZ	Plasma	0.5 M KH₂PO₄ (pH 4.0)-acetonitrile-acetic acid (65:35:0.1, v:v:v)	254	CLZ: 7.5	10–2,500	CLZ: 5	CLZ: 10	[50]
	CLZ and NCLZ	Plasma		254	NCLZ: 6.5	10–2,500	NCLZ: 5	NCLZ: 10	[50]
Phenomenex Gemini Phenyl Hexyl 110 A column (250 mm × 4.6 mm, 5 µm)	CLZ, NCLZ, olanzapine (OLZ), quetiapine, and several beta-blockers	Serum	Gradient elution of acetonitrile and potassium dihydrogen phosphate buffer (PB) pH 3.1 (containing 10 % methanol)	215, 226, 242, 299	CLZ: 30.39	5–3,200	CLZ: 1.12	CLZ: 5	[42]
		Serum		215, 226, 242, 299	NCLZ: 28.23	5–3,200	NCLZ: 3.3	NCLZ: 14.65	[42]
ODSIII column (250 mm × 4.6 mm ID, 5 μm)	CLZ, chlorpromazine (CPZ), and thioridazine	Plasma and wastewater	Acetonitrile, 0.05 M PB pH 3.0, methanol (42:35:23)	260	CLZ: 8	5–3,000	1.5	CLZ: 5	[12]
C18 ODS Hypersil reversed phase material (250 × 4.6 mm ID, 5 µm)	OLZ, CLZ, and demethylated metabolites	Serum	Acetonitrile–water–tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5	254	CLZ: 8.25 NCLZ: 6.36	10–800	—	10–20	[8]
Luna Omega 3 µm column (LC Column 150 × 3 mm)	OLZ, quetiapine, risperidone, aripiprazole, and CLZ	Saliva	Gradient eluent of water in formic acid and acetonitrile	240	CLZ: 16 NCLZ: 15	10–1,000	—	—	[23]
Waters C18 column (150 mm × 4.6 mm, 3.5 µm)	CLZ, NCLZ, clozapine-N-oxide (CNO)	Plasma	Acetonitrile and 62.4 mM PB (40:60, v/v) at pH 4.5, containing 0.3 % (v/v) triethylamine	220	CLZ: 4.1	CLZ: 100–2,000	CLZ: 23.6	CLZ: 71.52	[51]
					NCLZ: 3.5	NCLZ: 100–1,200	NCLZ: 19.3	NCLZ: 58.51
					CNO: 8.3	CNO: 100–1,000	CNO: 23.6	CNO: 71.43
Microsorb C18 reversed phase column (150 mm × 4.6 mm ID, 5 µm)	CLZ, NCLZ, and CNO	Plasma	Acetonitrile, methanol, and 10.4 mM pH 1.9 PB (17.5: 20: 62.5, v/v/v)	254	CLZ: 4.5	CLZ: 2.5–150	CLZ: 0.3	CLZ: 0.8	[52]
					NCLZ: 5.6	NCLZ: 2.5–150	NCLZ: 0.3	NCLZ: 0.7
					CNO: 7.8	CNO: 1.2–7.5	CNO: 0.6	CNO: 1.2
Whatman Partisil 10 ODS-3 column (250 mm × 4.6 mm ID, 10 µm)	CLZ and NCLZ	Plasma	Acetonitrile-methanol-buffered aqueous solution containing 5.0 g of dibasic phosphate adjusted to pH 4.0 with phosphoric acid (24: 12:64, v/v).	230	CLZ: 9.3	50–800	CLZ: 2	—	[5]
	CLZ and NCLZ	Plasma		230	NCLZ: 7.9	50–800	NCLZ: 1	—	[5]
Lichrospher 100 RP-18 endcapped column (11.9 × 4.6 mm, 5 µm)	CLZ, NCLZ, and CNO	Plasma	Acetonitrile-buffered aqueous solution containing 146 L of triethylamine and 200 L 85% phosphoric acid adjusted to pH 3.3	215	CLZ: 6.54	100–1,600	—	CLZ: 12.5	[7]
					NCLZ: 5.33			NCLZ: 10
					CNO: 7.45			CNO: 12.5
Kromasil Ultrabase C18 analytical column (250 mm × 4.6 mm ID, 5 µm)	CLZ, NCLZ, and CNO	Plasma and red blood cells	Acetonitrile-PB pH 7.0 (48–52, v/v)	254	CLZ: 11	—	CLZ: 10	CLZ: 20	[53]
					NCLZ: 4.9		NCLZ: 10	NCLZ: 30
					CNO: 2.8		CNO: 20	CNO: 20

3.3 Liquid chromatography-mass spectrometry (LC-MS)

LC methods are also commonly coupled with MS to achieve the benefits [30–32]. Recent advances of CLZ analysis have placed emphasis on the usage of tandem mass spectrometry, in which two different types of MS separation are used (Table 4). The analytes in the sample are thus separated twice based on their mass-to-charge ratio to greatly improve resolution.

Table 3

Summary of GC-MS methods of CLZ quantification

Column	Analyte	Matrices	Carrier	Flow rate (mL·min⁻¹)	Retention (min)	Linearity (ng·mL⁻¹)	LOD (ng·mL⁻¹)	LOQ (ng·mL⁻¹)	Ref.
Capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) with 5% phenylmethylsiloxane (HP-5MS)	CPZ, CLZ, haloperidol (HAL), OLZ, quetiapine, cyamemazine, and, levomepromazine	Plasma and oral fluid	Helium	0.8	9.15	Plasma: 20–600	Plasma: 10	Plasma: 20 Saliva: 10	[25]
		Plasma and oral fluid	Helium	0.8	9.15	Saliva: 10–400	Saliva: 5	Plasma: 20 Saliva: 10	[25]
Capillary column (30 m × 0.25 mm ID, 0.25μm film thickness) with 5% phenylmethylsiloxane (HP-5MS)	CPZ, levomepromazine, cyamemazine, CLZ, HAL, and quetiapine	Saliva	Helium	0.8	11.91	10–400	—	10	[10]
Equity 5 capillary column (30 m × 0.25 mm ID, 0.25μm film thickness) with 5% diphenyl/95% dimethylsiloxane	CLZ, bupropion, mirtazapine, sertraline, clomipramine and citalopram	Plasma, serum, and whole blood	Helium	1.5	13.45	25–800	7.5	25	[27]
Capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) with 5% phenylmethylsiloxane (HP-5MS)	CLZ, NCLZ	Plasma	Helium	1.0	CLZ: 19.5 NCLZ: 25.4	600	CLZ: 0.45 NCLZ: 1.59	CLZ: 1.37 NCLZ: 4.8	[45]
15 m wide-bore Heliflex Drug Three capillary column	CLZ	Plasma and serum	Nitrogen	14	6.04	100–3,000	—	35	[9]
Capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) with 5% phenylmethylsiloxane (HP-5MS)	CPZ, HAL, cyamemazine, quetiapine, CLZ, OLZ, and levomepromazine	Plasma	Helium	0.8	13.72	1–1,000	0.6	1.0	[24]

3.4 Electrochemical methods

Electrochemical methods of drug quantification rely on passing the current through a set of electrodes, which allows the drug compounds to accumulate and subsequently quantified [33,34]. Traditionally, it is uncommon for electrochemical methods to be applied to the quantification of drugs, especially in the clinical setting. However, a distinct advantage of this method is the highly customizable electrodes which can be designed to fit the purposes of an analysis. In recent years, several studies have been published which make use of specifically modified electrodes for the quantification of CLZ [15,16,35]. These studies have achieved degrees of sensitivity and specificity that are comparable to conventional chromatographic methods, as shown in Table 5.

Table 4

Summary of LC-MS methods of clozapine quantification

Column	Mobile phase	Wavelength (nm)	Flow rate (mL·min⁻¹)	Retention time (min)	LOD (ng·mL⁻¹)	LOQ (ng·mL⁻¹)	Ref.
Raptor™ biphenyl guard column (5.0 mm × 3.0 mm ID, 2.7 μm)	CLZ and NCLZ	Plasma	Gradient eluent of 0.1% (v/v) formic acid in deionized water and 0.1% (v/v) formic acid in acetonitrile:methanol (1:1)	CLZ: 12.4	—	CLZ: 10.0	[30]
Raptor™ biphenyl guard column (5.0 mm × 3.0 mm ID, 2.7 μm)	CLZ and NCLZ	Plasma		NCLZ: 11.4	—	NCLZ: 10.0	[30]
ACQUITY UPLC^® HSS T3, (2.1 mm × 100 mm ID, 1.8 µm)	Quetiapine, CLZ, and mirtazapine	Blood and tissue	Gradient elution of 0.1% HCOOH and 100% methanol	—	16–1,960	CLZ: 0.33	[54]
MACHEREY-NAGEL C18 (2.0 mm × 125 mm, 3 µm)	CLZ, OLZ, risperidone, and quetiapine	Plasma	Water (formic acid: 2.70 mmol·L⁻¹, ammonium acetate: 10 mmol·L⁻¹)–acetonitrile (53:47)	CLZ: 7.59	20–2,000	CLZ: 1.8	[6]
Restricted access material column consisting of porous octadecylsilane particles covered with bovine serum albumin (C18-BSA)	OLZ, quetiapine, CLZ, HAL, and CPZ	Plasma	Gradient elution of ammonium acetate solution and acetonitrile	CLZ: 5.01	—	—	[55]
KINTEX C18 column (50 mm × 3.0 mm ID, 5 μm)	Aripiprazole, CLZ, HAL, OLZ, paliperidone, quetiapine, risperidone, and ziprasidone	Serum	Gradient elution of aqueous ammonium formate and methanol	CLZ: 1.58	10–1,000	—	[31]
XBridge analytical column (2.1 mm × 100 mm ID, 3 μm)	CLZ, HAL, levomepromazine, OLZ, and quetiapine	Nails and hair	Gradient elution of ammonium formate with 0.1% FA (A) and ACN (B)	CLZ: 3	CLZ: 5–10,000 pg·mg⁻¹	CLZ: 2.5 pg·mg⁻¹	[56]
Synergi Polar RP column (150 mm × 2 mm ID, 4 μm)	CLZ, NCLZ, and CNO	Serum and urine	Gradient elution of 1 mM ammonium formate and methanol	CLZ: 8 NCLZ: 7.9 CNO: 8.9	2	CLZ, NCLZ, CNO (serum): 1	[32]
Synergi Polar RP column (150 mm × 2 mm ID, 4 μm)	CLZ, NCLZ, and CNO	Serum and urine	Gradient elution of 1 mM ammonium formate and methanol	CLZ: 8 NCLZ: 7.9 CNO: 8.9	2	CLZ, NCLZ, CNO (urine): 2	[32]
Ultrasphere cyano column (250 mm × 4.6 mm ID, 5 μm)	CLZ, NCLZ, CNO	Plasma	Ammonium acetate (60 mM, pH 7, not adjusted), methanol and acetonitrile (5:45:50, v/v/v)	CLZ, NCLZ, CNO: <4	1–1,000	CLZ, NCLZ, CNO: 1	[57]

3.5 Spectrophotometric methods

Spectrophotometry refers to the quantitative analysis of a material’s characteristic properties to absorb and emit different wavelengths of light. Spectrophotometry methods are widely available and applicable to the analysis of inorganic compounds, such as to determine the concentration of metallic ions in a particular material [17,19]. However, the use of spectrophotometry has also been extended to the application of drug analysis. Commonly, specific reagents are used to react with the analyte, which forms a coloured product that can be analysed in the spectrophotometer. The absorbance of the product is assumed to be proportional to the concentration of analyte in the sample. These methods are generally simple, inexpensive, and does not require high-end analytical instruments to be carried out [18,36,37]. The methods of CLZ quantification using spectrophotometric methods are presented in Table 6.

Table 5

Summary of electrochemical methods of CLZ quantification

Analyte	Method	Indicator electrode	Matrices	Solvent	Linearity (ng·mL⁻¹)	LOD (ng·mL⁻¹)	LOQ (ng·mL⁻¹)	Ref.
CLZ	Differential pulse voltammetry (DPV)	Glassy carbon electrode	Spiked human urine and serum	0.2 M PB at pH 6.0	100–100,000	8	—	[15]
CLZ and thioridazine	DPV	Carbon paste electrode	CLZ in HNO₃	Britton–Robinson buffer	500–45,000	61	—	[35]
CLZ	Linear sweep voltammetry	PPY modified GCE	Spiked human serum	Britton–Robinson buffer	10–5,000	3	—	[58]
Dopamine and CLZ	Cyclic voltammetry (CV), DPV	Screen printed carbon electrode	Spiked rat brain serum	—	500–2,092,000	97.6	—	[59]
Dopamine and CLZ	Cyclic voltammetry (CV), DPV	Screen printed carbon electrode	Spiked rat brain serum	—	10,000–910,000	97.6	—	[59]
CLZ	Voltammetry	Carbon ionic liquid electrode	CLZ in HCL	Distilled water and HCl pH 3.5	1–100	0.208	0.695	[60]
CLZ	DPV	Glassy carbon electrode	Spiked human serum	Phosphate buffered solutions (PBSs) pH 7	3–70	1.53	—	[47]
CLZ	CV, DPV	Carbon paste electrode	Spiked human serum	PB and acetate buffer (AB)	0.03–0.04	0.009	—	[48]
CLZ	CV, DPV	Carbon paste electrode	Spiked human serum	PB and acetate buffer (AB)	4–10,000	0.009	—	[48]
CLZ	CV	Glassy carbon electrode	Spiked serum and urine	PBS pH 8	50–375	1.67	—	[61]
CLZ	DPV	Graphite pencil lead	Spiked human plasma	0.01 m solution of K₄Fe	3–1,500	0.9	3.0	[62]
CLZ	CV, DPV, Chronoamperometry	Nanoparticles-modified screen-printed electrode	Spiked human urine	PBS (pH 7.0)	200–500,000 nM	70 nmol	—	[16]
CLZ	CV	Gold electrode modified with 16-mercaptohexadecanoic acid	CLZ in ethanol	0.05 mol l−1 TRIS–HCL (pH 8.1) buffer solution	1–50 nmol·mL⁻¹	0.007 nmol·L⁻¹	—	[63]
CLZ	CV	Glassy carbon electrode	—	AB (pH 5.50)	3–10 nmol·mL⁻¹	0.4 nmol·mL⁻¹	1.3 nmol·mL⁻¹	[64]
CLZ	DPV	Nanocomposite modified screen printed electrode	CLZ in Phosphate-buffered Saline	—	100–700,000 nM	30 nmol	—	[65]
CLZ	Square wave voltammetry	Glassy carbon electrode	Spiked human plasma and serum	0.1 M PB (pH 7)	100–2,000, 2,000–150,000 nM	30 nmol	100 nmol	[46]

4 Discussion

Half a century after its discovery, CLZ remains as a cornerstone in the management of schizophrenia despite various new antipsychotic medications coming into the market. This is mainly attributed to CLZ’s superior efficacy in the management of TRS and its low incidence of causing extrapyramidal symptoms [38,39]. A review of recent literature shows that LC methods are still widely used in the quantification of CLZ. However, a clear shift from traditional HPLC to LC-MS analyses can be observed [30–32]. The benefits afforded by MS detection greatly improves the utility and value of drug analysis. Apart from providing greater sensitivity and specificity, LC-MS studies are also quicker to be carried out. This maximizes the efficiency and throughput when a high demand of TDM is required in the clinical setting. One of the limitations of LC-MS is the high cost of the analytical instrument, which may limit the applicability of such methods in lower income regions. Additionally, HPLC and LC-MS methods are generally capable of simultaneous quantification of many drugs in a single sample [40–42]. This is especially relevant in psychiatric patients, which often require more than one medication to manage their conditions. These advantages may be the reason that HPLC and LC-MS remain the most widely used methods for the quantification of CLZ.

GC methods of CLZ analysis remain as an alternative to LC, despite the challenges in thermal decomposition of CLZ. Newer GC-MS methods, such as ones described by da Fonseca et al. [24] and Boumba et al. [27], were able to simultaneously quantify several antipsychotic drugs in the plasma, with a high degree of sensitivity and specificity. From the literature, it was observed that GC methods are more versatile in the types of matrices that they can be applied to. Studies done by Rosado et al. [25] and Caramelo et al. [10] described GC method of CLZ quantification in the saliva. This simplifies the sampling process and may be relevant in patients that are non-compliant to blood sampling. However, the degree of sensitivity and specificity in GC-MS are not superior to LC-MS methods, based on the results of literature reviewed. Furthermore, CLZ is almost fully metabolized in vivo and only trace amounts of the drug is excreted unchanged. Various cytochrome P450 enzymes are involved in CLZ metabolism, such as CYP1A2, CYP2D6, and CYP3A4. Through N-demethylation, CLZ is reduced to norclozapine (NCLZ) while N-oxidation of CLZ yields clozapine-N-oxide (CNO). Pharmacological testing of these two major metabolites has shown that NCLZ has limited activity, while CNO is inactive. In vitro, CLZ has also been shown to thermally decompose into NCLZ and CNO, particularly in GC-MS systems [43,44 Incidentally, 13 out of the 20 HPLC and LC methods that were included in this review reported data on CLZ metabolites. Meanwhile, the study published by Vardakou et al. [45] was the only GC method to report on CLZ metabolites. Among the electrochemical and spectrophotometric methods of CLZ quantification, no papers have reported any quantitative data on CLZ metabolites. While the quantification of CLZ metabolites may not be directly relevant in the clinical setting as they contribute very little to the drug’s efficacy, the data obtained from the quantification of the metabolites may provide a better understanding of the pharmacokinetics of the drug, particularly when pharmacogenomics research is involved.

Electrochemical methods of drug analysis are an emerging field of research that has seen numerous publications in recent years. The primary advantage of these methods is the high versatility of the electrodes used. Common working electrodes such as glassy carbon electrode and carbon paste electrode can be modified with a large variety of nanoparticles [15,35,46]. This allows the design of an electrode that best fits the purpose of the analysis. Several methods of voltammetry are also commonly employed, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry. Electrochemical methods also provide a good degree of sensitivity and specificity, and at the same time require only small sample volumes. However, current methods are largely restricted to the analysis of a single drug entity, and metabolites of CLZ are not able to be accurately detected [47,48].

Several spectrophotometric methods of CLZ quantification have also been described in recent years. These methods rely on the addition of reagents to form coloured complexes, which can then be quantified using a spectrophotometer. Due to challenges in human sample preparation as well as lower sensitivity and specificity, current methods of spectrophotometry are more suited for the quantification and quality control of pharmaceutical dosage forms. However, Eldidamony et al. [37] and Darwish et al. [19] have described spectrophotometric methods for the quantification of CLZ in human matrices such as plasma and urine.

5 Conclusion

The review of recent literature provides insight to the common methods of CLZ quantification, including chromatographic, electrochemical, and spectrophotometric analysis. It is seen that HPLC and LC-MS methods remain the most widely used and researched method of drug analysis. Recent advances in the field are highlighted, and it is observed that the analysis of CLZ has extended beyond conventional blood matrices to apply to nails, hair, and saliva as well. It may be beneficial to carry out further research on drug quantification using these novel matrices. This can provide clinicians with more options of drug sampling when blood sampling in a patient is not ideal.

Table 6

Summary of spectrophotometric methods of CLZ quantification

Analyte	Matrices	Solvent/reagent	Excitation (nm)	Linearity (μg·mL⁻¹)	Ref.
CLZ	Pure and dosage forms	3-methyl-2-benzothiazolinone hydrazone hydrochloride, p-n,n-dimethylphenylenediamine dihydrochloride, chloranilic acid	570, 690, 540	2–25, 10–120, 15–300	[17]
CLZ	Dosage forms	Potassium bromate in a perchloric acid	308	0.2–12	[36]
CLZ	Tablets and urine	Iodine, 7,7,8,8-tetracyanoquinondimethane, 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone, tetracyanoethane, p-chloranilic acid	365, 843, 460, 414, and 520	4–200	[19]
CLZ	Dosage forms	N-bromosuccinimide, acetic acid mixed anhydride reagent	320, 319	5.0–70.0, 8.0–24.0	[18]
Aripiprazole, clozapine, sulpiride	tablets, blood, urine	Bromophenol blue and bromothymol blue	408 and 406	1–11, 1–7	[37]

Funding information: UCSI Research Excellence & Innovation Grant (REIG-FPS-2020/064).
Author contributions: Jia Le Lim: writing – original draft, writing – review and editing, methodology, and formal analysis; Mogana Rajagopal: writing – review and editing, project administration, funding acquisition, and supervision; Gabriel Akyirem Akowuah: resources, visualization, supervision, and project administration; Fazlollah Keshavarzi: conceptualization, writing – review and editing, project administration, and supervision.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

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Received: 2023-06-27

Revised: 2023-10-06

Accepted: 2023-11-01

Published Online: 2024-03-21

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/revac-2023-0066

Keywords for this article

clozapine; scoping review; analysis; quantification; chromatography

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