New Trends in the Methodologies of Determination of Benzodiazepine Residues in Biological Samples

Abstract

The benzodiazepines are essential drugs used in medicine for anxiolytic, sedative, and hypnotic effects. According to the World Health Organization, the benzodiazepines are the most prescribed hypnotic drugs in the last decade (2010 at time), and their inappropriate use can damage the environment and human health. The availability of efficient analytical methods is crucial for the determination of these drugs in a complex matrix such as biological samples in clinical settings. In the last decade, several methods have been developed and have been applied to the detection and determination of benzodiazepines or their derivates. The present manuscript reviews selective and sensitive methodologies based on chromatographic, electrophoretic, and electrochemical systems for the determination of benzodiazepines in biological samples, covering the time of the last years and providing detailed information on sample pretreatment and instrumental conditions.

1. Introduction

Benzodiazepines (BDZs) are a group of psychoactive substances introduced in the 1960s. Benzodiazepines derive their chemical structure (Figure 1a) from the combination of a benzene ring with a diazepine ring [1,2,3]. A wide range of drugs belong to this group. In the human body, the BDZs act upon the central nervous system via the GABAA receptor (an ion channel comprised of five different subunits two α, two β, and one γ) [4]. The BDZ utilizes the α and γ subunits of the GABAA receptor and induces some effects through the central nervous system, including anxiolytic, antiseizure, hypnotic, amnestic, and muscle relaxant effects [4].

Figure 1. (a) General structure of BDZs; (b) chemical structures of several BDZs.

The type of BDZ depends on the components present as substituents in the diazepam ring, for example, Figure 1b shows several BDZ structures and the difference in substituents are categorized as sedative, anxiolytic, anticonvulsant, and hypnotic drugs [4,5,6]. According to the World Health Organization (WHO), BDZs are essential drugs used in the treatment of many central nervous system diseases such as depression, phobias, panic, aggressiveness, anxiety, insomnia, and epileptic attacks [1,3,6,7,8,9,10]. The advantages of using these drugs include efficiency, a fast start to action, a minor number of collateral effects, and minimum toxicity. These characteristics have allowed BDZs to be the psychotropic drugs most currently prescribed in the world, particularly in Western countries [8].

However, despite its advantages, BDZs can lead to negative health effects. Some studies attributed the drugs’ abuse to cognitive and sensory impairment, impaired psychomotor skills, and development of ataxia and hypotonia. Additionally, studies have reported that aggression and anxiety episodes can occur, non-melanoma cancer can develop, or even death [11,12]. For these reasons, in recent years, it has been necessary to apply rapid and accurate methodologies for the determination of benzodiazepines. The present work focuses on reviewing the applications of methodologies based on chromatographic, electrochemical, and electrophoretic systems in biological samples over the last few decades.

2. Methodology

The present review was conducted using literature from scientific databases in the last decades (2010–present), focusing on “Determination of benzodiazepines” as the primary keyword. The references were selected considering analytical techniques (liquid chromatography, gas chromatography, electrophoresis, and electrochemical techniques were included in the present work) to determine benzodiazepines in biological samples.

3. Pretreatment Samples

Sample pretreatment is a fundamental part of analysis in biological samples; the appropriate procedure influences the reproducibility and accuracy of analysis. The principal methodologies by pretreatment samples are solid phase extraction (SPE), liquid–liquid extraction (LLE), dispersive solid phase extraction (DSPE), and dispersive liquid–liquid extraction (DLLE) with their miniaturized versions (solid phase microextraction (SPME), dispersive solid phase microextraction (DSPME), liquid–liquid microextraction (LLME), and dispersive liquid–liquid microextraction (DLLME) [13,14,15,16].

SPE is based on the distribution of analytes between two phases (a liquid or donator phase and a solid or acceptor phase), where the sample (donator phase) is passed through adsorbent material (acceptor phase) to which the analytes have more affinity than the donator phase; later, the analytes are reextracted by elution with a specific solvent [13,14]. The principal advantage of SPE is the several sorbent materials that can be used such as fused-silica and carbonaceous or polymeric materials. Pretreatment sample methodologies based on SPE have been employed to extract several analytes in biological samples such as plasma, hair, blood, tissue, urine, and seminal fluid [13,14].

On the other hand, LLE is a common technique for pretreatment samples. LLE involves the use of two immiscible solvents: water (donator phase) and an organic solvent (acceptor phase), where the analytes partition between two liquid phases. Subsequently, the acceptor phase is evaporated and reconstituted in an appropriate solvent. In recent years, the methodologies based on LLE have been developed using ionic liquids and eco-friendly solvents, achieving acceptable results in the pretreatment of several samples for the determination of drugs, pesticides, metals, and other analytes [15,16].

4. Methods of Chromatography

Chromatographic methods are separation techniques that rely on the distribution of one or more analytes between a stationary phase and a mobile phase. The separation process occurs through interactions according to the different stationary phases and the analytes’ chemical properties. The procedure allows for the separation of one or more compounds [17]. Complex matrices are processed using coupled sample pretreatments [18,19,20,21,22,23,24,25,26,27,28,29,30].

4.1. Liquid Chromatography

Liquid chromatographic techniques, including rapid resolution liquid chromatography (RRLC), ultra performance liquid chromatography (UPLC), and high-performance liquid chromatography (HPLC), have been developed for the analysis of BDZs, metabolites, abused drugs, and psychoactive substances. Table 1 presents an overview of the application of liquid chromatography techniques for the determination of BDZs residues in biological samples [18,19,20,21,22,23,24,25,26,27,28,29,30].

Table 1. Liquid chromatography applications for the determination of BDZs in biological samples.

Analyte	Matrix	Sample Preparation	Instrumental Conditions	Limit of Detection	Recovery (%)	Ref.
Benzodiazepinas and metabolites (34 analytes)	Urine samples	Urine sample (1.0 mL) + internal standards (ISTD: zolpidem-d6 and prazepam-d5: 2 ng mL−1) was diluted 10-fold with deionized water.	Rapid resolution liquid chromatography (RRLC) Stationary phase: (set at 40 °C), Zorbax SB-C8/Hypersil GOLD perfluorophenyl (PFP). Mobile phase: (A) 0.1% formic acid/1 mM ammonium formate, and (B) acetronitrile/0.1% formic acid/1 mM ammonium formate (0.3 mL min−1). Gradient: 0–3 min (10% B), 3–5 min (20–40%), 5–9 min (40–70%). RRLC/triple quadruple MS (QqQ-MS): spray voltage (4000 V). Heated N2 gas (10 L min−1, 350 °C)	0.01–0.5 ng mL−1	80.2–98.5	[18]
Diazepam, oxazepam, temazepam, nordazepam, desalkylflurazepam, alprazolam, and α-hydroxyalprazolam	Urine samples (drivers: suspect, under influence)	Amounts: 500 μL of sample + 55 μL of MeOH (prepared in duplicate), fortificated with 55 μL spiked solution + 20 μL (2500 ng mL−1 internal standard) added to each tube. At the mixture was added 500 μL (β-glucuronidase), mixed (60 °C, 2 h.), centrifuged at 2500 rpm for 5 min (25 °C). A 200 μL aliquot of supernatant + 800 μL (10% ACN) vortexed.	UPLC Stationary phase: (set at 40 °C), Shim-pack XR ODS Mobile phase: binary gradient system: phase A (Ammonium formate/formic acid buffer), phase B (ammonium formate/formic acid buffer in ACN), (0.8 mL min−1). Gradient: The binary gradient, 0–1 min (5% B), 1–1.5 min (5–10% B), 1.5–2.5 min (10–25% B), 2.5–6.5 min (25–30% B), 6.5–8.5 min (30–35% B), 8.5–10 min (35–90% B), 10–12.5 (90–5% B), 12.5–15 min (5% B).	20 ng mL−1	90.8–108.6	[19]
Chlordiazepoxide, alprazolam, and lorazepam	Water, blood, and urine	Air-assisted liquid–liquid microextraction (AALLME) Amounts of 10 mL of samples (200 μg L−1 of each drug) were placed into a 15-mL centrifuge tube with conical bottom. Under the optimum condition (chloroform volume: 300 μL, pH of sample: 7, 10% w/v salt, extractions: 12 cycles, centrifugation rate: 4000 rpm, 9 min).	HPLC Stationary phase: C18 Mobile phase: ACN: PB (1 mL min−1.) Isocratic: ACN: PB (50 mM, pH = 3): THF (46:53:1, v/v/v).	0.7–2.9 μg L−1	81.2–92.1	[20]
Bromazepam, medazepam, and midazolam	Tablets, ampoules, capsules, serum samples	The serum solution was extracted (SPE), using C18 cartridge, activated (3 mL MeOH, 3 mL Water), apply the serum solution, and elution (10 mL MeOH-ACN; 1:1). The eluate solution was dried for 15 min, reconstituted with mobile phase.	HPLC-UV–Vis (240 nm) Stationary phase: C18 (50 °C). Mobile phase: ACN, MeOH, ammonium acetate (1.3 mL min−1). Isocratic: ACN, MeOH, and 0.05 M ammonium acetate (25:45:30, v/v/v), pH = 9.0.	1.02–3.03 μg mL−1	91.5–99.0	[21]
Clonazepam, lorazepam, alprazolam, and diazepam	Hair, urine, and wastewater	[PMIM]Br@TiO2 nanocomposite reinforced hollow fiber solid/liquid phase microextraction 1-pentyl-3-methylimidazolium bromide (ionic liquid) coated titanium dioxide ([PMIM]Br@TiO2) as membrane extraction, in two-phase supported: aqueous (donor phase), and octanol/nano [PMIM]Br@TiO2 (acceptor phase) by direct immersion sampling mode. Amount: 10 mL aqueous sample (pH = 7), 0.1 μg mL−1 analytes, 0.750 g NaCl was shacked at 1000 rpm for 1.0 min. The HF-SLPME fiber was immersed into the sample solution, and shacked (500 rpm, 45 min). After extraction, the analytes were desorbed with MeOH in ultrasonic bath for 10 min; 50 μL of the sample was injected.	HPLC-UV–Vis (230 nm) Stationary phase: C18. Mobile phase: MeOH, ACN (1.0 mL min−1) Isocratic: MeOH-ACN pure water optimized on (45:20:35 v/v).	0.08–0.5 ng mg−1	44.8–107.9	[22]
Bromazepam, clonazepam, alprazolam, nordiazepam, and diazepam	Plasma Samples	MIP fibers: template (0.1 mmol, DIA), monomer (0.4 mmol MAA), cross-linker (2.0 mmol, EGDMA), initiator (0.03 mmol ACVA, porogen solvent (3.0 mL chloroform), mixed 5.0 min, N2 atmosphere, 75 °C, 24 h, in capillary tubes. RAMIP fibers: MIP fibers were coated with 10.0 mL of 1% BSA, 0.05 M PB pH 6 (20 min), 10.0 mL 25% glutaraldehyde (5 h), 10.0 mL 1% sodium borohydride (20 min), washed and dried at 60 °C for 24 h. Samples: plasma samples (diluted in water 1.0:0.5 v:v), 1.0 mL of diluted sample, a RAMIP fiber was dipped inside it and shaken for 20 min at 500 rpm. The analyte was eluted (200 μL ACN, 5 min, 500 rpm.	HPLC-DAD (230 nm) Stationary phase: C18. Mobile phase: water, ACN, MeOH (1.3 mL min−1) Isocratic: water, ACN, MeOH (60:30:10, v/v/v).	5.0–30.0 μg L−1	NR	[23]
Triazolam, midazolam, and Diazepam	Urine	ZIF-8@Dt-COOH: 1.68 g glutaric anhydride, 120 mL DMF containing 3.48 mL APTES (-aminopropyltriethoxysilane), was stirred (3 h, 30 ° C). Subsequently, 2.0 g Dt (Diatomite), 100 mL of mixture of DMF, and 9 mL H2O was added. The resultant solution was stirred (5 h, 30 ° C). The Dt-COOH particles were washed (water, EtOH), dried at 25 °C. SPE cartridges: ZIF-8@Dt-COOH(2x) adsorbents (150 mg). Preconditioned (2 mL MeOH and water); 8 mL of sample (urine: H2O, 3:1, v/v) was passed onto the cartridge (0.4 mL min−1), washed (4 mL of NaH2PO4, 25 mM, pH = 5) and eluted (4 mL MeOH), dried under N2 gas stream at 35 °C, and reconstituted with a 0.2 mL (MeOH: H2O, 65:35, v/v).	HPLC-DAD (228 nm) Stationary phase: C18. Mobile phase: water, MeOH: B, (1.0 mL min−1). Gradient: 0–9 min (62–70% B), 9–11 min (70–70% B).	0.3–0.4 ng mL−1	80.0–98.7	[24]
Cannabinoids, opiates, amphetamines, cocaine, benzodiazepines, and methadone	Human serum, urine and, post-mortem blood	Amount: 100 μL sample, 10 μL deuterated internal standard. Protein precipitation (1000 μL ACN, stirred 15 min). The organic layer mixed with 10 μL HCl (0.1 M)-2-propanol, evaporated to dryness, and reconstituted in 100 μL of mobile phase.	HPLC-DAD (228 nm) Stationary phase: C18. Mobile phase: water, MeOH (0.2 mL min−1). Isocratic: (a) water, MeOH (95:5, v/v), (b) water, MeOH (3:97, v/v) with 10 mM ammonium acetate and 0.1% acetic acid.	0.01–3.6 ng L−1	85.0–113.0	[25]
Bromazepam, clonazepam, diazepam, lorazepam, and nordiazepam	Drugs in Human Plasma	Human plasma was treated by LLE (MeOH), using n-hexane-chloroform (70:30, v/v). At 200 µL of plasma, the protein was removed with 1000 µL of MeOH (mixed 30 s, centrifugation 5 min, 5000 rpm), the supernatant was dried to 50 µL volume (N2 Atmosphere, 30 ° C), adding 100 µL of the mobile phase.	HPLC-DAD (214 nm) Stationary phase: C18. Mobile phase: MeOH, PB (pH = 7.0, 20 mM) (1.0 mL min−1). Isocratic: MeOH, PB (50:50 v/v).	1.78–7.65 ng mL−1	95.2–107.5	[26]
Benzodiazepines, zolpidem, and their metabolites	Urine	An amount of 1 mL of urine was centrifuged (50,000× g, 3 min). Aliquots (120 µL) were mixed in 80 mL mixed with deuterium in the working solution. 5 µL of sample solutions were directly analyzed.	LC-MS/MS Stationary phase: C18. Mobile phase: water, ACN (2 mM ammonium trifluoroacetate and 0.2% acetic acid), (1.0 mL min−1). Gradient: 0–0.5 min (20% B), 0.5–9 min (20–95% B), 9–13 min (95–20%).	0.5–400 ng mL−1	63.0–104.6	[27]
11 benzodiazepines	Urine	Amounts: 170 µL internal solution, 30 µL ß-glucuronidase, 50 µL urine, mixed 10 s (≈25 °C), centrifuged at 4350 rpm 5 min and transferred to the auto sampler for injection of 5 µL.	LC-MS/MS Stationary phase: C18 Mobile phase: Mobile phase A (0.1% formic acid), Mobil phase B (ACN), (0.3 mL min−1). Gradient: 0–0.2 min (3% B), 0.2–0.5 min (3–20% B), 0.5–0.7 min (20–25% B), 0.7–0.95 min (25–25% B), 0.95–1.24 min (25–30% B), 1.24–1.49 min (30–30% B), 1.49–1.9 min (30–40% B), 1.9–1.94 min (40–99% B), 1.94–1.99 min (99–3% B), 1.99–3.0 min (3% B).	1–10 ng L−1	89.2–113.0	[28]
Drugs, benzodiazepines, and psychoactive substances	Urine samples	LLE: 1 mL urine samples + 0.5 mL 1.5 M CO32− electrolyte (pH 9.5) + 3 mL ethylacetate, mixed for 30 min. Centrifugation for 3 min at 2330 g, decanted the supernatant, was dried under N2. The residues were redissolved in 0.5 mL of 5% ACN with 0.1% formic acid, mixed 10 s, and filtered; 50 µL was injected into the LC-MS/MS system.	LC-MS/MS Stationary phase: ACE5 C18 column. Mobile phase: (A) 5% ACN-0.1% formic acid, and (B) 95% ACN-0.1% formic acid (0.8 mL min−1). Gradient (A): 0–1 min (100%), 1–5 min (100–0%), 5–17 min (0%), 17–17.1 (0–100%), 17.1–22 min (100%).	0.01–15.6 ng mL−1	70.3–120.6	[29]
9 benzodiazepines	Human serum	LLE: 500 μL (spiked serum), +300 μL of butyl acetate were mixed 5 min, centrifugated 14,500 rpm, 5 min. Each sample was maintained at −20 °C for 30 min. The organic phase (200 μL) was collected and evaporated under N2 atmosphere. The residues were dissolved in 50 μL of standard solution in mobile phase; 5 μL of each reconstituted extract was separated and analyzed by UHPLC-UHR-TOF MS. Blood samples (100 healthy volunteers).	UHPLC-MS Stationary phase: C18. Mobile phase: H2O/ACN 99:1, 2 mM ammonium formate, 0.1% formic acid. Gradient 1% B (0 min, 1 min), 70% B (5 min, 0.2-min), and 1% B (6.3 min, 0.7 min); the total run time was 7 min.	0.10–0.15 ng mL−1	84.4–99.2	[30]

RRLC has been used to analyze 34 substances in diluted urine samples, including BDZs, their metabolites, and analogous compounds such as zopiclone, zolpidem, and zaleplon. The RRLC was compared to LC-MS/MS with satisfactory results in terms of accuracy, precision, limits of detection (LODs) ranging from 0.01 ng mL⁻¹ to 0.5 ng mL⁻¹, and % recovery ranging from 80.2% to 98.5% [18].

In 2017, Dunlop et al. utilized an atmospheric pressure chemical ionization liquid chromatography/mass spectrometry (APCI-LC/MS/MS) to analyze seven BDZs (diazepam, oxazepam, temazepam, nordiazepam, desalkylflurazepam, alprazolam, and α-hydroxyalprazolam) in drivers who might have been under the influence of drugs for law enforcement purposes. The analysis entails the breakdown of conjugated BDZs using β-glucuronidase in an acetate electrolyte solution for a duration of 2 h at a temperature of 60 °C. The method was developed and validated in terms of matrix effect, accuracy (ranging from 90.82 to 108.65%), and precision with %RSD (Relative Standard Deviation) values of 10% for one day and 15% for different days. The linear range of the methodology was from 20 to 500 ng mL⁻¹. The approach was utilized to analyze 480 cases with positive samples for alprazolam (35%), oxazepam, nordiazepam, or temazepam (70%) [19].

Microextraction techniques, such as hollow fiber solid–liquid phase microextraction, have been used to determine alprazolam, clonazepam, diazepam, and lorazepam in complex matrices such as hair, urine, and wastewater by HPLC [20,21,22]. Eshaghi et al. utilized a membrane extraction technique using 1-pentyl-3-methylimidazolium bromide-coated titanium dioxide ([PMIM]Br@TiO2) for sample treatment. This approach demonstrated simplicity and effectiveness in comparison with traditional methodologies. According to the reports from the author, the fiber is employed once in order to minimize the possibility of cross-contamination, hence guaranteeing LODs ranging from 0.08 to 0.5 ng mL⁻¹. Patients undergoing therapy with BDZs were found to have positive samples of clonazepam, lorazepam, alprazolam, and diazepam in hair samples; the concentrations of these substances were measured to be 6.05, 7.34, 7.23, and 6.59, respectively, with a %RSD of less than 10.0 [22].

In 2019, a new type of Molecularly Imprinted Polymer (MIP) designate as Restricted Access Molecularly Imprinted Polymers (RAMIPs) was described. The RAMIPs are designed, synthesized, characterized, and applied as a fiber in SPME. The fiber was synthesized employing diazepam as a template molecule, methacrylic acid (MAA) as a functional monomer, and bovine serum albumin (BSA) as a cross-linking agent [23]. Carvalho et al. describe the use of BSA as a protective barrier against proteins in the analytical matrix. This improved the selectivity of the approach, resulting in a 98% exclusion capacity. The method’s validation was determined in a plasma sample, with LODs from 5.0 to 30 µg L⁻¹ [23].

Du, L. et al., describe the use of SPE coupled with HPLC for the analysis of three benzodiazepines (triazolam, midazolam, and diazepam) in urine samples. The adsorbent consists of diatomite-supported zeolite imidazolate framework-8 (ZIF-8@Dt-COOH). The contact modes between the analytes and the adsorbent are hydrophobic and π-π, based on their chemical composition. The development approach ensures LODs ranging from 0.3 to 0.4 ng ml⁻¹, with %recoveries ranging from 80.0 to 98.7% in spiked samples. The analysis of samples with values ranging from 10.9 to 21.7 ng mL⁻¹ for midazolam proved its feasibility [24].

Traditional sample preparation methods, such as LLE, have been successfully used to treat human plasma samples, providing a straightforward and dependable approach. The protein removal is carried out by utilizing 1 mL of methanol (MeOH). Afterwards, 50 µL of the supernatant is mixed with 100 µL of mobile phase and analyzed using RPLC/DAD under optimal conditions. The approach ensures minimum detectable amounts ranging from 1.78 to 5.59 ng mL⁻¹ (standard samples), and 4.16 to 6.34 ng mL⁻¹ (plasma). The recoveries range from 96.5% to 107.5%, and the precision, expressed as the %RSD, is less than 4.0% [26].

In liquid chromatography, the principal difference in methodologies was the pretreatment of the sample, highlighting the use of SPE with materials such as MIP, RAMIPs, and zeolites in the separation of BDZs, with apolar stationary phases, and the mobile phase consisted of mixtures of formic acid, ammonium formate, acetonitrile (ACN), phosphate buffer (PB), water, and MeOH in different proportions. In contrast, the spectroscopy detectors (UV–Vis, DAD) provide a LODS of 0.01 ng mL⁻¹ at 30.00 μg L⁻¹; however, the mass detectors (MS, MS/MS) provide a LODS of 0.01 ng mL⁻¹ at 400.00 ng mL⁻¹ in biological samples [18,19,20,21,22,23,24,25,26,27,28,29,30].

4.2. Gas Chromatography

Other chromatographic methodologies used in the analysis of BDZs include gas chromatography (GC). This technique requires that the analytes be transformed into the gaseous phase or volatile derivatives that can be separated and analyzed in the gaseous phase [17]. Table 2 shows an overview of the methodologies used for the determination of BDZs by GC [31,32,33,34,35,36,37,38,39].

Table 2. Gas chromatography applications for the determination of BDZs in biological samples.

Analyte	Matrix	Sample Preparation	Instrumental Conditions	Limit of Detection	Recovery (%)	Ref.
9 Benzodiazepines	Pericardial fluid	Pericardial fluid samples (500 μL) + 20 μL of the IS: internal standard (nordiazepam-D5 1 μg mL−1 and oxazepam-D5 10 μg mL−1) + 500 μL of PO43− buffer pH 4.1 mM were mixed. Peripheral blood samples (500 μL) + 20 μL of internal standards + 500 μL of PO43− buffer pH 9, 1 mM. SPE cartridges: conditioned (2 mL MeOH and 2 mL PO43− buffer pH 6, 1 mM), washed (3 mL MeOH/H2O 5:95, v/v, and 3 mL 0,3 M NH4, elution (3 mL Chloroform/Isopropanol 4:1, v/v), evaporated (N2 atmosphere, 40 °C), dissolved in 30 μL of derivatizant (N-tert-Butyldimethylsilyl-N-methyl-trifluoroacetamide with 1% of tert-Butyldimethylclorosilane) at 70 °C.	GC-MS/MS Stationary phase: HP-5MS column (30 m × 250 μm i.d. 0.35 μm thick film of 5%- (phenyl)methylpolisiloxane). Injector temperature: 250 °C. Gradient temperature: 140 °C (0 min, 1 min), and 290 °C (20 min). Mass detector: 300 °C, the ion source: 230 °C and the quadrupole at 150 °C.	0.002–0.1 µg mL−1	100.0	[31]
9 Benzodiazepines and zolpidem	Human urine and blood	Supramolecular solvent (SUPRASs) extraction method Urine and blood pretreatment (two healthy volunteers) 0.5 mL blood or 1.0 mL urine + 10 μL of IS, 1.0 mL THF and 200 μL 1-hexanol mixed for 1 min, centrifugated (12,000 rpm, 5 min). Each eluate was evaporated to dryness, and reconstituted with 50 μL of MeOH, and analyzed by GC-MS/MS.	GC-MS/MS Stationary phase: HP-5MS column (30 m × 250 μm i.d. 0.35 μm thick film). Mobile phase: helium gas (2.25 mL min−1). Injector temperature: 250 °C. Gradient temperature: 100 °C (1.5 min), 280 °C (23.7 min). Sample volume; 1.0 μL.	0.06–1.50 ng mL−1 Urine 0.30–15 ng mL−1 Blood	81.1–102.8 Urine 80.7–95.8 Blood	[32]
Benzodiazepines and their metabolites	Urine samples	Hollow-fiber liquid-phase microextraction (LPME) Hydrolysis: 100 µL of sodium acetate 2.0 M buffer solution (pH 4.5), 25 µL of β-glucuronidase enzyme (incubated, 90 min, 55 °C). LPME fiber (9 cm): mixture of dihexyl ether: 1-nonanol (9:1). 3.0 M of HCl introduced into the fiber (acceptor phase). After hydrolysis, the fiber was alkalinized urine (pH 10) with 10% NaCl. Samples: 2 mL urine shaking at 2400 rpm, 90 min. The acceptor phase was washed, dried and the residue derivatized with trifluoroacetic anhydride (TFAA) (10 min, 60 °C), +N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (1% tert-butyldimethylchlorosilane (MTBSTFA), 45 min at 90 °C.	GC-MS/MS Stationary phase: HP-5MS column (30 m × 250 μm i.d. 100 μm thick film). Mobile phase: helium gas (1 mL min−1). Injector temperature: 260 and 280 °C. Gradient temperature: 150 °C (1.0 min), 220 °C (30 °C min−1, hold 1 min) and 300 °C (30 °C min−1, hold 3 min), time analysis of 11.33 min. MS electron ionization: 70 eV.	0.1–15 ng mL−1	3.3–92.7	[33]
Alpha-hydroxyalprazolam, oxazepam, lorazepam, nordiazepam, and temazepam	Urine samples	1 mL urine + 0.100 mL of IS (100 ng mL−1 of AHAL-d5, OXAZ-d5 (used for OXAZ and LORA), NORD-d5 and TEMA-d5) + 2 mL 0.1 M acetate buffer (pH 4.75) + 0.050 mL β-glucuronidase (type HP-2), mixed and incubated 60 min, 55 °C. Cooled was centrifuged 3000 rpm, 5 min. SPE CEREX® CLIN II cartridges (3 mL), were activated: 1 mL CO32− buffer (pH 9), 1 mL of H2O/ACN (80:20), and 1 mL H2O. Elution: 1 mL CH2Cl2/MeOH/NH4 (85:10:2), and evaporated to dryness at 55 °C. Derivatization: 0.050 mL of ethyl acetate + 0.050 mL of MTBSTFA (w/1% MTBDMCS) were added to the dried extracts (vortexed and incubated 20 min, 65 °C). LC-MS-MS 0.5 mL urine + 0.100 mL IS + 1 mL 0.1 M acetate buffer (pH 4.75) + 0.025 mL of β-glucuronidase (type HP-2) was mixed and incubated for 60 min at 55 °C. The cooled mixture was centrifuged 3,000 rpm, 5 min. Samples were transferred to 3 mL UCT Clean Screen® XCEL I cartridges, and washed (1 mL methylene chloride). Analytes were eluted with 1 mL ethyl acetate/NH4 (100:2), evaporated to dryness at 55 °C. Samples were reconstituted with 20 µL (mobile phase).	GC-MS/MS Stationary phase: HP-ULTRA 1 (15 M, 0.20 mm, 0.33 μm) column. Mobile phase: helium gas (f 0.9 mL min−1). Injector temperature: No reported. Gradient temperature: No reported. Sample volume: 0.5 μL. LC-MS/MS Stationary phase: C18 (1.7 μm, 2.1 × 50 mm). Mobile phase: (A) 0.1% formic acid and (B) ACN. Sample volume: 10 μL.	5.53–19.31 ng mL−1 GC-MS 1.96–15.83 ng mL−1 LC-MS/MS	98.3–102.5	[34]
Diazepam, midazolam, flurazepam, and alprazolam	Water, urine, and plasma	DLLME Amount: 5 mL sample solution + 1 mL acetone as disperser solvent containing 150 µL of n-dodecane/BA (2:1 v/v) as extraction solvent (2 min vortex). The BDZs were extracted into fine droplets of extraction solvent. The mixture was centrifuged, 5 min, 4000 rpm. The dispersed fine droplets were collected on the upper of aqueous phase (140 µL) + 10 µL ACN (mixed 30 s), the analytes were transferred to small volume of ACN. After centrifugation, the ACN was collected (10 µL); 2 µL was injected.	GC-microelectron capture detector (GC-mECD) Stationary phase: HP-5 column (30 m × 320 μm i.d. 250 μm thick film). Mobile phase: helium gas (flow rate of GC-mECD; 2 mL min−1 and GC-MS/MS 1 mL min−1). Injector temperature: 220 °C. Gradient temperature: GC-mECD; 50 °C (0 min), 220 °C (20 °C min−1) and 290 °C (15 °C min−1, hold 3 min), GC-MS/MS; 50 °C (2 min), 280 °C (25 °C min−1, hold 25 min). MS electron ionization: 70 eV. Sample volume: 2 mL.	0.01–1.00 mg L−1	3.2–88.6	[35]
Diazepam, midazolam, and alprazolam	Ultra-pure water, tap water, fruit juices, and urine samples	SPE-DLLME Supelclean LC-C18 (6 mL, 500 mg). Activated (3.0 mL acetone, 3.0 mL H2O, flow 1.0 mL min−1); 60 mL spiked sample (pH 10.0), was passed through SPE (7.5 mL/min). Wash step (2.0 mL H2O), elution (2.0 mL of acetone) reduced to 0.5 mL by evaporation (N2 atmosphere). The residue was disperser solvent in DLLME procedure. Ultra-pure water (pH 11.0: 4.5 mL) + 0.5 mL eluent + 40.0 µL chloroform was injected into the aqueous solution with a 5.00 mL gas-tight syringe obtaining a cloudy solution. The BZPs were extracted into the droplets of chloroform. The emulsion was centrifuged (5000 rpm, 5 min), the chloroform phase was sedimented; 2.0 µL of the settled phase was injected into the GC-FID system for analysis.	GC-MS/MS/GC-FID Stationary phase: HP-5 column (30 m × 320 μm i.d. 250 μm thick film). Mobile phase: helium gas (3 mL min−1). Injector temperature: 180 °C. Gradient temperature: 180 °C (2 min), 290 °C (12 °C min−1). MS electron ionization: 70 eV. Sample volume; 2 mL.	0.02–0.10 µg L−1	92.5–110.5	[36]
Phenazepam, diclazepam, flubromazepam, and etizolam	Urine	Ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction (UA-LDS-DLLME) Ethyl acetate (168 μL, lidocaine; IS, 5 μg mL−1) + 1.0 mL urine samples (pH 11.3; 165 μL ethyl acetate, mixed 5.5 min). The cloudy solution, was centrifugated (10,000 rpm, 3 min), the supernatant was injected (GCQQQ-MS).	GC-triplequadrupole mass spectrometry (GC-QQQ-MS) Stationary phase: DB-5MS capillary column (30 m × 320 μm i.d. 250 μm thick film). Mobile phase: helium gas (1.2 mL min−1). Injector temperature: 260 °C. Gradient temperature: 100 °C (2 min), 220 °C (20 °C min−1) and increased to 300 °C (10 °C min−1). Solvent delay time was 3.5 min. MS electron ionization: 70 eV. Sample volume: 1 μL.	1.00–3.00 ng mL−1	81.4–91.3	[37]
Venlafaxine, mirtazapine, olanzapine, paroxetine, and sertraline	Postmortem samples	Pericardial fluid was centrifuged (14,000 rpm, 5 min), 0.3 mL were used for analysis, and spiked with Proadifen (SKF) (20 μL Sol. 10 μg mL−1), diluted (1.1 mL) in H2O and 150 mg of NaCl in a DLLME. Optimal conditions were: 175 µL of chloroform, 750 µL ACN. The mixture was centrifuged, and the droplet formed was collected by a 100 μL syringe. The organic solvent was evaporated (N2 atmosphere) at 40 °C, dried and redissolved with 40 μL of MeOH, injected (2 μL aliquot).	Electron ionization for GC-MS Stationary phase: HP5-MS capillary column (30 m × 320 μm i.d. 250 μm thick film). Mobile phase: helium gas (1.0 mL min−1). Injector temperature: 250 °C. Gradient temperature: 100 °C (1 min), 220 °C (35 °C min−1, hold 1 min), 260 °C (8 °C min−1, hold 2 min), 280 °C (5 °C min−1, hold 3 min), 290 °C (5 °C min−1, hold 5 min). A time analysis 24.43 min. MS electron ionization: 70 eV. Sample volume: 1 μL. MSD: 300 °C, the ion source at 230 °C, and the quadrupole at 150 °C.	0.005–0.20 µg mL−1	85.0–105.0	[38]
15 benzodiazepines	Blood samples	HPLC grade water (2.0 mL at pH 7.0) + 20 µL (fludiazepam, oxazepam-d5, clonazepam-d4, and 7-aminoclonazepam-d4 (200 ng mL−1 in MeOH) added to 0.2 mL of blood sample. After acidification (0.15 mL of 1.5 M HCl), was mixed, incubated 2 min and centrifuged (3500 rpm, 5 min), the supernatant was subjected to SPE. SPE (Oasis MCX cartridge); activated (1.0 mL MeOH, 1.0 mL 0.1 M HCl pH 1.0, 1.0 mL min−1). The sample was passed at SPE system at 1.0 mL min−1, washed three times with 1.5 mL of 0.1 M HCl at pH 1.0; 1.5 mL of 1-PrOH, water at 0.15 M HCl mixture (60:40 v/v) and 1.0 mL ACN. SPE cartridge was dried for 2 min, eluted with 2.0 mL of 5% NH4 in MeOH, evaporated to dryness (35 °C, N2). The residue was silylated with 100 µL of MTBSTFA/ACN/ethyl acetate (20:40:40 v/v/v) at 85 °C for 30 min. After a derivatization process (25 °C), 1.0 µL was injected.	Gas chromatography–negative-ion chemical ionization mass spectrometry (GC-NICI-MS) Stationary phase DB-5HT capillary column (30 m × 320 μm i.d. 100 μm thick film). Mobile phase: methane (2.5 mL min−1)/helium gas (3.5 mL min−1). NICI-MS: voltage (625 ± 50 V), emission: 49 ± 1 µA, electron energy: 149 ± 1 eV, repeller: 2.8 ± 0.2 V and ion focus: 130 ± 2 V. Injector temperature: 250 °C. Gradient temperature: 180 °C, increased to 325 °C (50 °C min−1, hold 1 min). A time analysis 3.9 min. Sample volume: 1 μL.	0.24–0.62 ng mL−1	90.3–107.8	[39]

Álvarez-Freire L et al. published a study in 2018 describing the implementation of an SPE method employing Bond Elut Certify cartridges for sample extraction. An investigation was conducted on blood and pericardial fluid samples to analyze nine benzodiazepines, namely diazepam, midazolam, nordiazepam, oxazepam, bromazepam, temazepam, lorazepam, alprazolam, and clonazepam. The LOD of the SPE-GC/MS approach was verified at a concentration of µg mL⁻¹. The precision and accuracy were determined by measuring inter-day and intra-day variations at three different concentration levels (0.05, 0.15, and 0.30 µg mL⁻¹). The %RSD was found to be less than 11.63%, with %recoveries ranging from 93.76 to 106.15%. The approach was utilized on 12 positive samples obtained from cases of both natural and suicide deaths. The results indicate considerable variations between the two samples for nordiazepam, with concentrations of 0.3 µg mL⁻¹ in pericardial fluid and 0.02 µg mL⁻¹ in blood. Similarly, there are significant variances for oxazepam, with concentrations of 0.24 µg mL⁻¹ in pericardial fluid and 0.18 µg mL⁻¹ in blood [31].

Supramolecular solvents and the LLE method were used to analyze nine benzodiazepines and a benzodiazepine analogue (zolpidem) in biological samples, specifically human urine and blood. The technique uses a supramolecular solvent (tetrahydrofuran/1-hexanol) to efficiently extract low-molecular-weight molecules, followed by their detection using GC-MS/MS. The SUPRASs-GC-MS/MS methodology provides LODs ranging from 0.30 to 1.50 ng mL⁻¹ [32]. The precision and accuracy were assessed in terms of inter- and intra-day repeatability of each analyte. In all cases, a %RSD of less than 10% was achieved, indicating high precision. Furthermore, the analysis of real blood samples yielded recovery rates ranging from 80.74% to 95.84%. The procedure was effectively utilized in two participants, from whose samples were obtained 24 h following the oral administration of diazepam and zolpidem. The collected samples were then examined, and the analytes were identified based on their retention times. The concentrations of diazepam in urine ranged from 1.98 to 49.19 ng mL⁻¹, whereas the quantities of zolpidem in blood ranged from 8.37 to 10.95 ng mL⁻¹ [32]. The supramolecular solvent that is created has the ability to efficiently extract low-molecular-weight chemicals from samples that contain water.

Hollow fibers have been applied in liquid phase microextraction (LPME) coupled with GC to analyze BDZs and their primary metabolites in urine samples. The LPME-GC method involves enzymatic hydrolysis, followed by extracting the analytes using a hollow fiber. The acceptor phase in the fiber is then removed, dried, and the residue is derivatized for subsequent GC analysis. The approach yields LODs ranging from 0.1 to 15 ng mL⁻¹. The intra-day precision was consistently below 111.5% in all cases, while the inter-day precision was below 20.5%. To verify the precision, spiked samples were analyzed providing %recovery ranging from 89.1% to 111.6%. The suggested method was used to test samples from volunteers who reported using benzodiazepines for medical purposes. The analysis revealed the presence of nordiazepam and oxazepam in quantities of 69.1 and 109.6 ng mL⁻¹, respectively [33].

In 2016, Perez et al. conducted a thorough comparison of GC-MS/MS and LC-MS/MS techniques for analyzing five benzodiazepines: alpha-hydroxyalprazolam, oxazepam, lorazepam, nordiazepam, and temazepam [34]. The LOD values obtained for GC-MS/MS ranged from 5.53 to 19.31 ng mL⁻¹, while for LC-MS/MS, they ranged from 1.96 to 15.83 ng mL⁻¹. The accuracy and precision of both methods were assessed at eight different levels (20, 40, 75, 100, 125, 200, 500, and 1000 ng mL⁻¹). A t-test was used to confirm the absence of significant differences in the findings obtained from both approaches. The efficacy of LC-MS/MS in the analysis of BDZs has been proven through statistical demonstration. This technique provides accurate and precise results while minimizing the need for sample preparation [34].

The technique of DLLME coupled to GC-QQQ-MS has been used to analyze four benzodiazepines (phenazepam, etizolam, flubromazepam, and diclazepam) in urine, using low-density solvents. The extraction process parameters were optimized using a Box–Behnken design. The best conditions for the pH of the sample solution, volume of the extracting solvent, and extraction time were determined to be pH 11.3, 165 μL of ethyl acetate, and a mixing duration of 5.5 min. The technique provides LODs ranging from 1 to 3 ng mL⁻¹. The approach demonstrates a %RSD (precision and accuracy.) < 5.9%. The method was employed to quantify the concentration of phenazepam, which was reported to be 158 ng mL⁻¹ [35].

Cation-exchange polymeric sorbents have been utilized in SPE, in conjunction with GC and negative-ion chemical ionization mass spectrometry, for the detection and quantification of fifteen benzodiazepines in human blood. The methodology involves collecting the blood serum, which is then passed through an SPE cartridge to clean up the sample and remove any unwanted substances. The analytes are then treated with a derivative and injected into the GC/NICI-MS system. The efficiency of the sample preparation was assessed based on the relative response factor [39]. The LODs provided by the method range from 0.24–0.62 ng mL⁻¹. The accuracy of the method was assessed by comparing the measured concentration in blood, obtained through calibration curves, with spiked samples of benzodiazepines in blood samples. The obtained values for accuracy range from 89.5% to 110.5% [39].

According to the literature, gas chromatography techniques for the determination of BDZs are used, the principal stationary phase is an HP-5 column (30 m × 320 μm i.d. 250 μm), and helium gas is used as a carrier gas at a flow rate of 1 mL min⁻¹ at 3 mL min⁻¹. DLLME is one of the most used sample pretreatments, highlighting the low solvent consumption that provides an eco-friendly analysis. In addition, the main detector system is mass detectors (MS, MS/MS) with LODs of 0.06–0.1 ng mL⁻¹ at 0.1 µg mL⁻¹ in biological samples such as serum, urine, blood, and pericardial fluid [31,32,33,34,35,36,37,38,39].

5. Capillary Electrophoresis

Capillary electrophoresis has been employed routinely in the determination of benzodiazepines such as clonazepam, tetrazepam, midazolam, and diazepam [17,40] due to the property of electromigration of these molecules. This technique allows the separation of ionic, polar, and nonpolar molecules, molecules with different chirality, and different molar masses. There are different electrophoretic techniques to separate benzodiazepines, such as capillary electrophoresis (CE), capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), non-aqueous capillary (NACE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), micellar electrokinetic capillary chromatography (MECC), and capillary electrochromatography (CEC) [40]. Table 3 shows the main electrophoresis methods used and the conditions of separation and determination [41,42,43,44,45,46]. CZE is limited in the study of benzodiazepines due to poor separation of these compounds caused by their similar hydrophobicity. To identify the behavior of benzodiazepines in CZE, Shiung et al. studied the separation of eight benzophenones with a phosphate-borate buffer in a pH range from 7.5 to 11.5 with an optimum separation of pH = 9.2 (25 °C and 20 kV). They describe the effect of the amount of OH groups in the benzophenone structure on the electrophoretic mobility that was observed. They concluded that with a higher amount of OH groups, the electrophoretic mobility increases due to the conjugation of the OH groups to the aromatic rings. However, the use of CZE in biological samples has been limited in recent years due to the properties [40].

Table 3. Determination of BDZs by capillary electrophoresis in biological samples.

Analyte	Matrix	Sample Preparation	Technique and Instrumental Conditions	Limit of Detection	Ref.
9 benzodiazepines	Human serum	Amounts of 100 μL of serum (blank or spiked) + 900 μL of 2 M TRIS/chloride buffer pH 9 + 1000 µL of 1-chlorobutane were mixed 1 min and then put into an ultrasonic bath for 5 min. The mixture was centrifuged at 5000 rpm for 5 min, samples were maintained at −80 °C for 10 min. The supernatant was collected and evaporated under N2 atmosphere at 50 °C. The residue was reconstructed in 100 μL of ACN and analyzed.	NACE Background electrolyte and separation conditions: 25 mM ammonium acetate with 100 mM trifluoroacetic acid in ACN, 25 °C and 20 kV. Internal standards: deuterated analogues (etizolam-d5, phenazepam-d4, and diazepam-d5). Detector: tandem mass spectroscopy.	1.5–15.0 ng mL−1	[41]
8 benzodiazepines	Human serum and hair	A mixture was made of 1 mL of serum or 45 mg hair + 1 mL borate buffer pH 9.5 + 3 mL of ethyl acetate, the mixture was treated by microwave-assisted extraction (5 min at 55 °C for serum and 15 min at 65 °C for hair), after the process the solution was centrifugated (10 min, 4 °C, 4000 rpm). Subsequently, the organic layer was collected and evaporated under N2 atmosphere at 40 °C. The residue was then dissolved in 100 μL of a mixture 1:10 background electrolyte: water, the solution was centrifuged 13,000 rpm, 5 min, for degassing and analyzed.	CE Background electrolyte and separation conditions: 100 mM formic acid and ACN (80:20, v/v), 25 °C and 30 kV. Internal standards: deuterated analogues of BZDs (0.1 mg mL−1). Detector: mass spectroscopy.	0.4–1.2 ng mL−1 for serum, 6.0–23.0 pg mg−1 for hair	[42]
5 benzodiazepines	Blood	Amount: 0.5 mL of blood + 1.5 mL of water + 20 μL of phosphoric acid and 10 μL of doxapram, the mixture was loaded onto the SPE cartridge (activated with 1 mL of MeOH and 1 mL of water). The cartridge was washed with 1 mL of a solution of 2% NH4OH and 5% MeOH. The analytes were eluted with 1 mL of ethyl acetate–MeOH (12:1), the eluate was a mixture with 20 μL of HCl (1%) and was evaporated under an air stream at 50 °C. Subsequently, the residue was redissolved with MeOH–water (1:4).	CE Background electrolyte and separation conditions: 150 mM PB (pH 2.4) with 20% MeOH, 25 °C, and 16 kV. Internal standard: doxapram 1 mg mL−1. Detector: UV (200 and 210 nm).	8.0–30.0 ng mL−1	[43]
8 benzodiazepines	Urine	Amounts of 3 mL of urine fortified with each analyte from 50 to 2000 ng mL−1 + 1 mL of EtOH + 500 µL of dichloromethane were spun in a vortex (60 s) and centrifugated 6000 rpm, 3 min. The lower phase was collected and evaporated to dryness at 40 °C. The residue was reconstituted with 500 µL of buffer, centrifugated at 13,000 rmp for 5 min, and analyzed.	MECC Background electrolyte and separation conditions: 30 mM SDS, 10 mM sodium tetraborate and 15% MeOH (pH 8.8), 25 °C, and 23 kV. Internal standard: nitrazepam (500 ng mL−1) Detector: photodiode array detection.	20.0–30.0 ng mL−1	[44]
Tetrazepam	Blood	Two drops of blood (25 µL each) were deposited on the FTA DMPK C DBS cards and dried for 1.5 h at room temperature. Afterward, one disc was cut from each deposited drop, the two discs + 1 mL of H2O + 1 mL of buffer (pH 13.5) + 3 mL of hexane isoamyl alcohol 99:1, the mixture was treated by microwave-assisted extraction (2.5 min at 55 °C). Subsequently, the mixture was centrifuged for 10 min at 4000 rpm(4 °C). The supernatant was separated and dried under N2 atmosphere at 40 °C. Finally, the residue was dissolved in 50 μL of background electrolyte.	CE Background electrolyte and separation conditions: 50 mM acetic acid and MeOH 60:40, 25 °C and 30 kV. Internal standard: zolpidem-D6 (150 ng mL−1) Detector: mass spectroscopy.	14.7 ng mL−1	[45]
Clonazepam	Plasma samples	The pH of plasma samples was adjusted with HCl 6 M. The sample was put into an ultrasound-assisted electromembrane extraction system, using a polypropylene hollow fiber with 2-nitrophenyl octyl ether as supported liquid membrane. The acceptor solution was analyzed by CE.	UA-EME-CE Background electrolyte and separation conditions: 100 mM PB (pH 2.0), at 25 °C and +18 kV. Detector: photodiode array detection (210 nm).	3.0 ng mL−1	[46]

On the other hand, Švidrnoch et al. performed the separation of nine benzodiazepines (bentazepam, pyrazolam, deschloroetizolam, flubromazepam, flubromazolam, nimetazepam, diclazepam, phenazepam, and etizolam) by NACE. Table 3 displays the separation conditions. In this investigation, a volatile nonaqueous electrolyte is based on 20 mM of ammonium acetate in ACN and 100 mM of trifluoroacetic acid. The separation and selectivity of this investigation was satisfactory with an LOD suitable for biological samples where diazepines can be found in the order of tens to hundreds of ng L⁻¹ [41].

Woźniakiewicz et al. studied the simultaneous determination by capillary electrophoresis of eight benzodiazepines (lorazepam, 7-aminoclonazepam, alprazolam, clonazepam, diazepam, 1-benzylpiperazine, estazolam, and tetrazepam) in human serum and hair samples. To perform each analysis, the samples were previously extracted with 1 mL of borate buffer (pH = 9.5) and 3 mL of ethyl acetate by microwave-assisted extraction, with a recovery of 88.6–113.4% for serum and 86.1–107.4% for hair. The separation was carried out using a mixture of 100 mM of formic acid and ACN for 20 min with a voltage over 30 kV in a polyamide-coated fused silica capillary. With these conditions, the separation had a good resolution, an LOD of 0.4–1.2 ng mL⁻¹ for serum, 6.0–23.0 pg mg⁻¹ for hair, and a limit of quantification (LOQ) of 1.3–4.1 ng mL⁻¹ for serum and 20.0–77.0 pg mg⁻¹ for hair [42].

Cui et al. studied the separation of five benzodiazepines (diazepam, midazolam, nordiazepam, flurazepam, and diazepam) in a mixture of forty-six drugs in human blood samples by CE. They performed an SPE to determine the drugs in blood. The separation was carried out at 25 °C with a running electrolyte of 150 mM phosphate (pH 2.4) with 20% MeOH with a UV detector (200 and 210 nm). The LODs were in the range of 8–30 ng mL⁻¹ being lower for nordiazepam and flurazepam. This determination allowed for the analysis of forensic samples and to determine the cause of death by different drugs [43].

Ole et al. proposed a method to adequately determine eight benzodiazepines (chlordiazepoxide, estazolam, temazepam, midazolam, clonazepam, medazepam, lorazepam, and lormetazepam) using electrokinetic capillary chromatography in urine samples. The analytes were previously extracted by D LLME. The best separation of BZDs was obtained with a running buffer composed of 30 mM SDS, 10 mM sodium tetraborate, and 15% MeOH (pH 8.8); a sample buffer composed of 10 mM SDS and 2 mM sodium tetraborate and a voltage of 23 kV were applied. The application of field-amplified sample stacking improves the analysis time and sensitivity of the method, as well as the performance the analysis on urine samples [44].

Świądro et al. developed a method to determine different types of drugs in blood samples collected in vivo or post-mortem by capillary mass-coupled electrophoresis combined with the dried blood spot (DBS/CE-MS) method. To perform the analysis, the samples were prepared using microwave-assisted extraction (MAE). Separation of the analytes was performed for 25 min using a voltage of +30 kV in a silica capillary. The temperature was set to 25 °C. The detection was carried out in the positive ion mode, and profile spectra were acquired in the mass range of 100–1450 m/z using a mass spectrometer. The analysis of the benzodiazepine tetrazepam showed an LOD of 14.7 ng mL⁻¹ with an LOQ of 49 ng mL⁻¹ with a matrix effect of 99.8% (n = 6) and a recovery of 99.5%. In addition to presenting a good LOD, LOQ, and recovery, this method has the advantage of using the DBS technique to guarantee the stability of the analytes for 14 days after sample collection (storage at 15 °C) [45].

On the other hand, Seyfinejad et al. [46] developed another method to determine chlordiazepoxide in human plasma using ultrasound-assisted electromembrane extraction coupled with capillary electrophoresis (UA-EME-CE) with photodiode array detection. Utilizing the optimal conditions (100 mM PB adjusted to pH 2.0, 25 °C and +18 kV) the UA-EME provided an extraction recovery of 58% in 13 min. Furthermore, this technique achieved a pre-concentration factor of 203 and a LOD of 3 ng mL⁻¹ with good repeatability.

Electrophoretic techniques for determination of BDZs are diverse (NACE, CE, and MECC); therefore, the pretreatment of sample and experimental conditions as background electrolyte and separation voltage are specific to each technique; however, by these techniques, the lower LODs were of 0.4 ng mL⁻¹ and 6 pg mg⁻¹ reported by Woźniakiewicz et al., and provided with a mass detector in serum and hair samples, respectively. This represents an important difference compared to spectroscopic detectors with LODs around a range from 3 to 20 ng mL⁻¹ in samples such as blood, plasma, and urine samples [41,42,43,44,45,46].

6. Electrochemical Methods

Electrochemical methods such as voltammetry or potentiometry have been recently studied in drugs such as diazepam, clonazepam, tetrazepam, and oxazepam. Recent potentiometric and voltammetric studies are reported in Table 4 [47,48,49,50,51,52,53,54,55,56].

Table 4. Determination of BDZs by electrochemical methods.

Analyte	Matrix	Technique and Electrode Electrolyte and Conditions	Limit of Detection	Ref.
Alprazolam, diazepam, clonazepam, oxazepam, and chlordiazepoxide	Plasma samples	Differential-pulse voltammetry Electrode: dopamine-polyfolic acid nanocomposite modified glassy carbon electrode. Electrolyte and conditions: solution of NaOH (0.1 M) supporting electrolyte, measurements step potential of 0.2 V, Epulse = 0.05 V, tpulse = 0.2 s, and scan rate = 50 mV s−1.	0.025–0.50 μM	[47]
Alprazolam, diazepam, clonazepam, oxazepam, and chlordiazepoxide	Plasma samples	Differential-pulse voltammetry Electrode: silver nanoparticle-nitrogen-doped graphene quantum dots sink modified gold electrode. Electrolyte and conditions: 0.1 M NaOH (supporting electrolyte), measurements step potential of 0.2 V, Epulse = 0.05 V, tpulse = 0.2 s, and scan rate = 50 mV s−1.	3.8–61.8 μM	[48]
Tetrazepam	Human Serum	Differential-pulse voltammetry Electrode: nitrogen-doped carbon nanoparticle-modified carbon paste electrode. Electrolyte and conditions: PB (pH 7.4, 0.1 M), scan rate of 25 mV s−1.	5.00 ng mL−1	[49]
Clonazepam	Urine and human serum	Differential-pulse voltammetry Electrode: silver nanofibers/ionic liquid nanocomposite-modified glassy carbon electrode. Electrolyte and conditions: PB (pH 7.0), scan rate of 50 mV s−1.	66.00 nM	[50]
Clonazepam	Human serum	Differential-pulse voltammetry Electrode: glassy carbon electrode modified with Fe3O4/R-SH/Pd nanocomposite. Electrolyte and conditions: PB (pH 7.0), pulse amplitude of 55 mV, pulse width of 40 ms, and scan rate of 80 mV s−1.	3.02 nM	[51]
Flunitrazepam	Plasma samples	Differential-pulse voltammetry Electrode: carbon paste electrode modified with MnFe2O4 and gold nanoparticles. Electrolyte and conditions: Britton–Robinson buffer solution 0.04 M, scan rate of 50 mV s−1.	0.33 μM	[52]
Olanzapine	Human serum	Potentiometry Electrode: carbon paste electrode modified with olanzapine-tungstophosphate. Electrolyte and conditions: acetate buffer solution (pH 5.5), temperature = 15–55 °C.	0.50 μM	[53]
Olanzapine, oxazepam, and lorazepam	Urine	Potentiometry Electrode: PVC-membrane ion-selective electrode. Electrolyte and conditions: acetate buffer solution (pH 4.5).	0.32–4.60 μM	[54]
Clonazepam	Human serum	Differential-pulse voltammetry Electrode: a screen-printed carbon electrode modified with Cu nanoparticles anchored on porous silicon. Electrolyte and conditions: Britton–Robinson buffer (0.1 M, pH 7.0), tpulse = modulation time: 100 ms and Epulse = modulation amplitude: 70 mV).	0.015 μM	[55]
Clonazepam	Human serum and urine	Differential pulse voltammetry Electrode: screen-printed carbon electrode modified with WS2 Nanorods and WS2 Nanoballs. Electrolyte and conditions: Britton–Robinson buffer (0.05 M, pH 7.0), amplitude of 0.05 V, pulse period of 0.2 s, and a pulse width of 0.05 s.	2.37 nM	[56]

Ashrafi et al. designed a polydopamine-polyfolic acid nanocomposite modified glassy carbon electrode for the determination of five benzodiazepines (alprazolam, diazepam, clonazepam, oxazepam, chlordiazepoxide) in human plasma [47]. Cyclicvoltammetry differential pulse voltammetry and square wave voltammetry methods were used to study the electrochemical behavior of the benzodiazepines. The most sensitive detection was obtained with the differential-pulse voltammetry (measurements step potential = 0.2 E_pulse = 0.05 V, t_pulse = 0.2 s and scan rate = 50 mV/s) with an electrolytic solution of 0.1 M NaOH. Under these conditions, the LOQ obtained was 0.040, 0.033, 0.50, 0.025, and 0.04 μM for alprazolam, diazepam, clonazepam, oxazepam, and chlordiazepoxide, respectively. The results indicate that polydopamine-polyfolic acid has an essential role in signal amplification. The same method was employed by modifying a gold electrode with silver nanoparticle-nitrogen-doped graphene quantum dots [48]. In this case, the LOQs were increased to 3.8, 61.8, 3.8, 7.7, and 61.8 μM in plasma for diazepam, clonazepam, oxazepam, chlordiazepoxide, respectively.

On the other hand, Ma et al. developed a modified carbon paste electrode with nitrogen-doped carbon nanoparticles for electrochemical detection of tetrazepam in human blood serum [49]. The differential-pulse voltammetry analysis shows that the electrode is sensitive and selective with an LOD of 5 ng mL⁻¹ and a broad linear range of 0–650 µg mL⁻¹. These results were compared with an ELISA technique. Khoshroo et al. [50] developed an electrochemical sensing platform based on nanostructured silver fibers and ionic liquid composites for an electrochemical sensor for the detection of clonazepam. The electrochemical analysis was carried out by differential pulse voltammetry in urine and human serum. This method offers a wide linear range from 0.1 to 250 μM with an LOD of 66 nM, and acceptable reproducibility and stability compared with the HPLC method.

For their part, Lofti et al. [51] carried out an electrochemical determination of the clonazepam drug based on a glassy carbon electrode modified with Fe₃O₄/R-SH/Pd nanocomposite by differential-pulse voltammetry. The synthesized nanocomposite consists of Fe₃O₄, 3-aminopropyltriethoxysilane, cyanuricchloride, 2-mercaptoethanol, and Pd. The optimal conditions were a pulse amplitude of 55 mV, a pulse width of 40 ms, and the scan rate of 80 mV/s with a PB solution (pH 7.0). With these conditions, the designed sensor has a linear range from 10 nM to1 μM with an LOD of 3.02 nM.

Asiabar et al. have determined flunitrazepam in human plasma with a differential-pulse voltammetry method. This investigation was carried out with a carbon paste electrode modified with MnFe₂O₄ and gold nanoparticles. At the optimal experimental conditions, the anodic current measured during the oxidation of the flunitrazepam at 0.41 V showed a linear response to the concentrations of the flunitrazepam in a range of 0.1–100 μM. This method has an LOD of 0.33 μM. The authors report that this method permits measuring flunitrazepam in human plasma samples in the presence of some organic compounds and mineral ions with an economic advantage.

Potentiometric methods were studied in human samples [53,54,55,56]. Rouhani and Soleymanpour [53] developed a potentiometric sensor fabricated for the analysis of olanzapine in real samples. The electrode design consists of a carbon paste electrode modified with an olanzapine–tungstophosphate ion pair. The modified electrode showed an LOD of 5 × 10⁻⁷ M with high selectivity. The electrode has excellent thermal stability in a temperature range of 15–55 °C. This method permits studying the interaction between olanzapine drug and β-cyclodextrin and determining olanzapine in human blood serum. Wassel and Abdullatif developed a potentiometric sensor with a PVC-membrane type to determinate olanzapine, oxazepam, and lorazepam in urine [54]. The membranes incorporate the ion associates of the drug cations with ammonium reineckate and later dispersed into 2-nitrophenyl-octylether or dibutyl sebacate. The inorganic or organic compounds studied did not present any interference in this study. The proposed method was applied to the determination of BDZs in urine, with results comparable to HPLC measurements.

Allahnouri et al. carried out a voltammetry determination of clonazepam with a screen-printed carbon electrode modified with copper nanoparticles anchored on porous silicon in spike human blood serum samples [55]. Under the optimum conditions (t_pulse = modulation time: 100 ms and E_pulse = modulation amplitude: 70 mV), the calibration is linear in the 0.05–7.6 μM clonazepam concentration range, and the LOD is 15 nM. The authors discuss that the method is reproducible, repeatable, highly selective, and sensitive. Chen et al. developed a screen-printed carbon electrode modified with WS2 and nanorods and WS2 nanoballs to determine clonazepam in biological samples (human serum and urine) [56]. The modifications made to the electrode increased the peak current and improved its ability to facilitate reactions compared to the unmodified electrode. These properties permit a linear response at 10–551 µM range, a lower LOD (2.37 nM), and high sensitivity. This electrode has excellent selectivity, including co-interfering compounds. Moreover, the electrode has good recovery (96.5% to 98.6%) for serum and urine samples.

7. Conclusions

Benzodiazepines are an important group of drugs prescribed for different health conditions. However, the misuse or excessive use of these drugs is a potential public health and environmental problem. For these reasons, in the last decades, the development of techniques and methodologies to determine these molecules has attracted attention. Therefore, this review covers different methods using chromatography, electrophoresis, and electrochemistry techniques for the determination of BDZs in biological samples. The different methodologies show acceptable LODs for the analytes, highlighting the use of mass detectors in chromatography and electrophoretic systems. Looking at the sample preparation techniques, electrochemical methodologies make use only of dilution of the original sample in an electrolyte solution, while the chromatographic and electrophoretic methodologies make use of SPE, SPME, among others, with different, innovative, and modern sorbent materials or modification of materials preexisting. On the other hand, other preparation techniques, such as LLME and LPME, employ environmentally friendly solvents. With respect to methodologies analyzed in the present work, the different techniques have different advantages and advantages such as minimal pretreatment (electrochemical techniques), multianalyte determination with lower LODs in samples (electrophoretic and chromatographic systems), demonstrating significant progress in the development of efficient and effective eco-friendly methodologies for the determination of BDZs and their derivatives in complex matrices.