LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram's effectiveness in the treatment of cocaine dependence. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).     

An LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol in rat liver tissues

Context: Salidroside and its metabolite p-tyrosol are two major phenols in the genus Rhodiola L. (Crassulaceae). They have been confirmed to possess various pharmacological properties and are used for the prophylaxis and therapeutics of many diseases. Several analytical methods have been developed for the determination of the two compounds in plant materials and biological plasma matrices. However, these methods are not optimal for biological samples containing complex organic interferences, such as liver and brain tissues.

Objective: This study aimed to further develop and validate a simple and specific LC–MS/MS method for the determination of salidroside and its metabolite p-tyrosol in rat liver tissues using paracetamol as the internal standard (IS).

Materials and methods: Salidroside and p-tyrosol with the IS paracetamol and liver tissues were used as model compounds and biological samples. Samples were processed by protein precipitation (PP) with methanol, the supernatant was dried under nitrogen and the residue was reconstituted in a mobile phase that consisted of a mixture of acetonitrile and water (1:9, v/v). Salidroside and p-tyrosol were detected in negative mode under multiple reaction monitoring (MRM) by a triple quadrupole tandem mass spectrometer coupled with electrospray ionization.

Results: Standard curves were linear over the concentration range of 50–2000?ng/mL with correlation coefficients of 0.995 or better for both salidroside and p-tyrosol. The intra- and inter-day accuracy for salidroside ranged between 104.90 and 112.73% with a precision of 3.51–14.27%. For p-tyrosol, the intra- and inter-day accuracy was between 92.38 and 100.59%, and the precision was 8.54% or less. The stability data showed that no significant degradation occurred under the experimental conditions. The recoveries were 111.44, 108.10, and 102.00% for salidroside at concentrations of 50, 500 and 2000?ng/mL, respectively, and were 105.44, 105.50, and 113.04% for tyrosol at concentrations of 50, 500 and 2000?ng/mL, respectively. The matrix effects were 83.85–92.45% for salidroside and 85.61–92.49% for p-tyrosol at three QC levels. This method was successfully applied to a liver tissue distribution study of salidroside and its metabolite p-tyrosol in rats.

Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for the intrinsic quality control of biological matrices, especially tissue samples.     

Reversed-phase HPLC/FD method for the quantitative analysis of the neurotoxin BMAA (β-N-methylamino-L-alanine) in cyanobacteria

A method has been developed and optimized in order to detect and quantify the non-protein amino acid β-N-methylamino-L-alanine(BMAA) in cyanobacteria. The novelty of the method is that we have used methanol instead of acetonitrile as the eluent. The method includes extraction with 0.1 M trichloroacetic acid (free BMAA) or protein hydrolysis with 6 M hydrochloric acid (total BMAA), derivatization with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and reversed-phase high-performance liquid chromatography analysis with fluorescence detection (HPLC/FD). Detection limits ranged from 0.35 to 0.75 pg injected, while quantification limits ranged from 1.10 to 2.55 pg injected for total and free BMAA hydrolysis, respectively. The linear response range was up to 850 pmol in both methods, embracing three orders of magnitude. The method was successfully applied to a lyophilized estuarine species of Nostoc (LEGE 06077). All previous published methods for BMAA quantification, using HPLC/FD, have reported the usage of acetonitrile. This is the first report using methanol as the mobile phase. Although the elution strength differs with both solvents, the final method proved efficient for the quantification of BMAA in this complex sample. The method resulted effective, low-priced, and simple, being suitable for routine monitoring of BMAA in cyanobacteria.     

An LC–MS/MS method for the simultaneous determination of chlorogenic acid,forsythiaside A and baicalin in rat plasma and its application to pharmacokinetic study of Shuang-huang-lian in rats

An LC–MS/MS method was developed for the simultaneous determination of chlorogenic acid, forsythiaside A and baicalin, three major ingredients in Shuang-huang-lian preparations, in rat plasma. Following extraction by methanol–ethyl acetate–trifluoroacetic acid (49:49:2, v/v/v), the extracted analytes were separated on a reverse phase C12 column using a gradient mobile phase system of acetonitrile–water containing 0.1% formic acid. The limits of quantification were between 1.0 and 2.1 ng/mL, the precision was <7% and the accuracy was between 94% and 107%. The validated method was applied to a comparative pharmacokinetic study in rats after administration of Shuang-huang-lian solutions via intravenous, peroral or intratracheal routes. The results showed that the three chemical markers were more rapidly and thoroughly absorbed following pulmonary delivery as compared with peroral administration.     

Validated LC–MS/MS method for the determination of sarpogrelate in human plasma: Application to a pharmacokinetic and bioequivalence study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C₁₈ column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3 → 135.3 and of I.S. at m/z 264.1 → 58.0. The assay was validated over the concentration range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 μL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were ≤6.4% and ≤5.4%, respectively, with accuracy (relative error, R.E.) in the range 0.5–3.6%. The method was successfully applied to a pharmacokinetic and bioequivalence study enrolling 22 Chinese volunteers administered sarpogrelate tablets.     

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.     

An accurate,rapid and sensitive determination of tramadol and its active metabolite O-desmethyltramadol in human plasma by LC–MS/MS

A rapid, sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the simultaneous determination of tramadol and its active metabolite, O-desmethyltramadol in human plasma is developed using propranolol as internal standard (IS). The analytes and IS were extracted from 200 μL aliquots of human plasma via protein precipitation using acetonitrile. Chromatographic separation was achieved in a run time of 2.0 min on an Aquasil C18 (100 mm × 2.1 mm, 5 μm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method was fully validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. A linear dynamic range was established from 1.0 to 600.0 ng/mL for tramadol and 0.5–300.0 ng/mL for O-desmethyltramadol. The method was successfully applied to a bioequivalence study of 200 mg tramadol tablet formulation in 27 healthy Indian male subjects under fasting condition.     

Development and validation of a highly sensitive LC–MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC–MS/MS). A minimal volume of human plasma sample (20 μL) was prepared by simple deproteinization with 80 μL of acetonitrile. Chromatographic separation was performed using Kinetex C₁₈ column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 → 242.17 for acacetin and m/z 277.59 → 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1–500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5–95.6 %. This validated LC–MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.     

UPLC–MS/MS for the determination of azilsartan in beagle dog plasma and its application in a pharmacokinetics study

The purpose of the study is to develop an ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) to determinate the concentration of azilsartan in the dog plasma. After precipitated by methanol, the plasma sample containing azilsartan and diazepam (internal standard, IS) was determined by UPLC–MS/MS. The mobile phase consisted of acetonitrile-water was pumped at a flow rate of 0.3 ml/min in gradient elution. Kinetex 2.6 μ XB-C18 column (50 × 2.1 mm, 100 Å; Phenomenex, USA) were used for LC separations. The column temperature was 30 °C and the injection volume was 5 μl. The electrospray ionization (ESI) and multiple reaction monitoring (MRM) were applied at the transitions of m/z 457 → 279 (azilsartan) and m/z 285 → 193 (diazepam), respectively. The developed method was identified a good linearity over a concentration range of 2.5–5000 ng/ml. The lower limit of quantitation (LLOQ) was 2.5 ng/ml. The intra-day and inter-day precision (relative standard deviation, RSD%) were less than 10% and accuracy (relative error, RE %) was less than 5% at three quality control levels. The extraction recovery of azilsartan at three quality control levels were 82.41 ± 0.68%, 98.66 ± 11.00%, 102.43 ± 0.82%. And the recovery for IS (100 ng/ml) was 91.75 ± 0.54%. A validated UPLC–MS/MS method was firstly developed for the quantification of azilsartan in dog plasma and it was applied to the pharmacokinetics study.     

Validated LC–MS/MS assay for the determination of felbinac: Application to a preclinical pharmacokinetics study of felbinac trometamol injection in rat

A rapid and sensitive analytical method based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of felbinac in rat plasma, bile, urine, feces and tissue. Sample preparation involved liquid–liquid extraction with ethyl ether–dichloromethane (60:40, v/v). Chromatography of felbinac and the internal standard probenecid was performed within 2 min on a Venusil MP C₁₈ column (100 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of acetonitrile–5 mM ammonium acetate containing 0.1% formic acid (pH 3.0) (80:20, v/v) at a flow rate of 1.2 ml/min. Detection by electrospray negative ionization mass spectrometry and multiple-reaction monitoring of the transitions of felbinac at m/z 211.1 → 167.0 and of probenecid at m/z 283.9 → 239.9 was linear over the concentration range 5–5000 ng/ml with a lower limit of quantitation of 5 ng/ml using a sample volume of only 50 μl. Intra- and inter-day precisions (as relative standard deviation, R.S.D.) were ≤7.3% and ≤6.4%, respectively, and accuracy (as relative error, R.E.) was in the range −2.1 to 7.4%. Recoveries and matrix effects were satisfactory in all the biological matrices examined. The method was applied to a preclinical pharmacokinetic study in rat involving a single intravenous injection of felbinac trometamol.     

Development and validation of a LC–MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for the identification and quantification of bivalirudin in human plasma using nafarelin as the internal standard. Following protein precipitation with methanol, the analytes were separated on a C₁₈ column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Quantification of bivalirudin was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 1091.4 → (356.4 + 227.4) for bivalirudin and m/z 662.4 → 328.5 for IS. The lower limit of quantification was 1.25 ng/ml, and the assay exhibited a linear range of 1.25–500 ng/ml. The developed assay method was successfully applied to a pharmacokinetic (PK) study in healthy volunteers after intravenous administration of bivalirudin.     

Validated LC–MS (ESI) assay for the simultaneous determination of amitriptyline and its metabolite nortriptyline in rat plasma: Application to a pharmacokinetic comparison

A rapid, sensitive and specific method based on high performance liquid chromatography with electrospray ionization mass spectrometry (HPLC–MS/ESI) has been developed for the simultaneous determination of amitriptyline and nortriptyline in rat plasma. Sample preparation involved liquid–liquid extraction with methyl t-butyl ether after alkalified with 0.5 mol/l NaOH. Chromatographic separation was performed on a XB-C4 column (4.6 mm × 250 mm, 5 μm, Welch Materials) with a mobile phase consisting of 10 mM ammonium acetate (0.6‰ formic acid)–acetonitrile (60:40, v/v) at a flow rate of 1.0 ml/min. Calibration curves were linear within the ranges of 10–3200 ng/ml for amitriptyline and 10–1000 ng/ml for nortriptyline. This method was successfully applied to the pharmacokinetic study in rats after intravenous injection of amitriptyline hydrochloride.     

Identification and characterization of a photolytic degradation product of telmisartan using LC–MS/TOF,LC–MS,LC–NMR and on-line H/D exchange mass studies

Telmisartan, an anti-hypertensive drug, was subjected to stress studies under ICH prescribed conditions of hydrolysis (acidic, neutral and basic), photolysis, oxidation and thermal stress. The drug showed labiality under only photo-acidic condition by forming a single degradation product. HPLC separation of the drug and the degradation product was achieved on C-8 column using gradient method. To characterize the product, a complete mass fragmentation pathway of the drug was initially established. Subsequently, the degradation product peak was subjected to LC–MS/TOF and on-line H/D exchange mass studies. Based on these studies, a tentative structure was assigned to the product as 3-((1,7′-dimethyl-2′-propyl-1H,3′H-2,5′-bibenzo[d]imidazol-3′-yl)methyl)-6H-benzo[c]chromen-6-one, which was verified through ¹H LC–NMR experiments.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

LC–MS/MS method for simultaneous analysis of cladrin and equol in rat plasma and its application in pharmacokinetics study of cladrin

A rapid and selective LC–MS/MS method for simultaneous analysis of cladrin and equol in female rat plasma has been developed and validated. The chromatographic separation was carried out on RP18 column, and MS/MS analysis was performed in triple quadrupole mass spectrometer with electrospray ionization. The method was linear for the concentration range from 7.8 to 1000 ng/ml for cladrin and 3.9 to 1000 ng/ml for equol. The intra-day and inter-day accuracy and precision of the method were within the acceptable limits. The validated LC–MS/MS method was successfully applied for the pharmacokinetics study of cladrin at 10 mg/kg in female S.D. rats.     

Using ambient mass spectrometry and LC–MS/MS for the rapid detection and identification of multiple illicit street drugs

In this study the recently developed technique of thermal desorption electrospray ionization/mass spectrometry (TD–ESI/MS) was applied to the rapid analysis of multiple controlled substances. With the reallocation of mass spectral resources [from a standard ESI source coupled with liquid chromatography (LC) to an ambient TD–ESI source], this direct-analysis technique allows the identification of a wider range of illicit drugs through a dual-working mode (pretreatment-free qualitative screening/conventional quantitative confirmation). Through 60-MRM (multiple reaction monitoring) analysis—in which the MS/MS process was programmed to sequentially scan 60 precursor ion/product ion transitions and, thereby, identify 30 compounds (two precursor/product ion transitions per compound)—of a four-component (drug) standard, the signal intensity ratios of each drug transition were comparable with those obtained through 8-MRM analysis, demonstrating the selectivity of TD–ESI/MS for the detection of multiple drugs. The consecutive analyses of tablets containing different active components occurred with no cross-contamination or interference from sample to sample, demonstrating the reliability of the TD–ESI/MS technique for rapid sampling (two samples min^?1). The active ingredients in seized drug materials could be detected even when they represented less than 2 mg g^?1 of the total sample weight, demonstrating the sensitivity of TD–ESI/MS. Combining the ability to rapidly identify multiple drugs with the “plug-and-play” design of the interchangeable ion source, TD–ESI/MS has great potential for use as a pretreatment-free qualitative screening tool for laboratories currently using LC–MS/MS techniques to analyze illicit drugs.     

LC–MS/MS method for the quantitation of decitabine and venetoclax in rat plasma after SPE: Application to pharmacokinetic study

This study developed a novel, sensitive and selective LC-MS/MS method for the concurrent determination of DCB and VTX in rat plasma using encorafenib as internal standard (IS). To identify DCB, VTX, and IS, the positive multiple reaction monitoring (MRM) mode was used. Chromatographic separation was carried out using a reversed-phase Agilent Eclipse plus C18 column (100 mm × 2.1 mm, 3.5 µm) and an isocratic mobile phase made up of water with 0.1% formic acid and acetonitrile (50:50, v/v, pH 3.2) at a flow rate of 0.30 mL/min for 3.0 min. Prior to analysis, the DCB and VTX with the IS were extracted from plasma using the solid-phase extraction (SPE) method. High recovery rates for DCB, VTX and IS were achieved using the C18 cartridge without interference from plasma endogenous. The developed method was validated as per the FDA guidelines over a linear concentration range in rat plasma from 5–3000 and 5–1000 ng/mL for DCB and VTX, respectively with r² ≥ 0.998. For both drugs, the lower limits of detection (LLOD) were 2.0 ng/mL. After the HLOQ sample was injected, less than 20% of the LLOQ of DCB, VTX, and less than 5% of the IS carry-over in the blank sample was attained. The overall recoveries of DCB and VTX from rat plasma were in the range of 90.68–97.56%, and the mean RSD of accuracy and precision results was ≤6.84%. For the first time, the newly developed approach was effectively used in a pharmacokinetic study on the simultaneous oral administration of DCB and VTX in rats that received 15.0 mg/kg of DCB and 100.0 mg/kg of VTX.     

Simultaneous determination of rupatadine and its metabolite desloratadine in human plasma by a sensitive LC–MS/MS method: Application to the pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of rupatadine and its metabolite desloratadine in human plasma. After the addition of diphenhydramine, the internal standard (IS), plasma samples were extracted with a mixture of methyl tert-butyl ether and n-hexane (1:1, v/v). The analysis was performed on a Ultimate™ AQ-C18 (4.6 mm × 100 mm, 5 μm) column using a mobile phase consisting of a 80/20 mixture of methanol/water containing 0.0005% formic acid pumped at 0.3 ml min⁻¹. The analytes and the IS were detected in positive ionization mode and monitoring their precursor → product ion combinations of m/z 416 → 309, 311 → 259, and 256 → 167, respectively, in multiple reaction monitoring mode. The linear ranges of the assay were 0.1–50 and 0.1–20 ng ml⁻¹ for rupatadine and desloratadine, respectively. The lower limits of reliable quantification for both rupatadine and desloratadine were 0.1 ng ml⁻¹, which offered high sensitivity and selectivity. The within- and between-run precision was less than 7.2%. The accuracy ranged from −9.2% to +6.4% and −7.2% to +7.2% for rupatadine and desloratadine in quality control samples at three levels, respectively. The method has been successfully applied to a pharmacokinetic study of rupatadine and its major metabolite after oral administration of 10, 20 and 40 mg rupatadine tablets to healthy Chinese volunteers.     

Development of a LC–MS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of antrodin B and antrodin C in rat plasma. Both target compounds, together with the internal standard (diazepam), were extracted from rat plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Agilent XDB-C₈ column with an isocratic mobile phase consisting of acetonitrile and water (70:30, V/V) at a flow rate of 0.5 mL/min. The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source operating in positive ionization mode. The assay exhibited a linear dynamic range of 47.6–4760 ng/mL for antrodin B and 56.6–5660 ng/mL for antrodin C. The intra- and inter-day precision was less than 5.3% and the accuracy was less than 2.7% for both analytes. The validated method has been applied to the pharmacokinetic study of antrodin B and antrodin C in rats following oral administration of Antrodia camphorata extract.     

Development and validation of an EI–GC–MS method for the determination of benzodiazepine drugs and their metabolites in blood: Applications in clinical and forensic toxicology

Benzodiazepines are used widely in daily clinical practice, due to their multiple pharmacological actions. The frequent problems associated with the wide use of benzodiazepines, as well as the multiple incidents of poisonings, led to the necessity for the development of a precise, sensitive and rapid method for the simultaneous determination of the 23 most commonly used benzodiazepines (diazepam, nordiazepam, oxazepam, bromazepam, alprazolam, lorazepam, medazepam, flurazepam, fludiazepam, tetrazepam, chlordiazepoxide, clobazam, midazolam, flunitrazepam, 7-amino-flunitrazepam, triazolam, prazepam, nimetazepam, nitrazepam, temazepam, lormetazepam, clonazepam, camazepam) in blood. A gas chromatographic method combined with mass spectrometric detection was developed, optimized and validated for the determination of the above substances. This method includes liquid–liquid extraction with chloroform at pH 9 and two stages of derivatization using tetramethylammonium hydroxide and propyliodide (propylation), as well as a mixture of triethylamine:propionic anhydride (propionylation). The recoveries were higher than 74% for all the benzodiazepines. The calibration curves were linear within the dynamic range of each benzodiazepine with a correlation coefficient higher than 0.9981. The limits of detection and quantification for each analyte were statistically calculated from the relative calibration curves. Accuracy and precision were also calculated and were found to be less than 8.5% and 11.1%, respectively. The developed method was successfully applied for the investigation of both forensic and clinical toxicological cases of accidental and suicidal poisoning.