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Clinical Chemistry 52: 1346-1355, 2006. First published May 18, 2006; 10.1373/clinchem.2005.065631
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(Clinical Chemistry. 2006;52:1346-1355.)
© 2006 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Liquid Chromatography–Tandem Mass Spectrometry for Detection of Low Concentrations of 21 Benzodiazepines, Metabolites, and Analogs in Urine: Method with Forensic Applications

Oscar Quintela1,2, François-Ludovic Sauvage1, Fabienne Charvier1, Jean-Michel Gaulier1, Gérard Lachâtre1 and Pierre Marquet1,a

1 Department of Pharmacology-Toxicology, Limoges University Hospital, Limoges, France.
2 Forensic Toxicology Department, Institute of Legal Medicine, University of Santiago de Compostela, C/San Francisco, Spain.

aAddress correspondence to this author at: Department of Pharmacology-Toxicology, Limoges University Hospital, 87042 Limoges, France. Fax 33-555-05-61-62; e-mail pierre.marquet{at}unilim.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Commonly used methods for detecting benzodiazepines (BZPs) and BZP-like substances, such as zolpidem and zopiclone, may not detect low concentrations of these drugs. We developed a liquid chromatographic–tandem mass spectrometric method for identifying these drugs and their relevant metabolites.

Methods: We extracted BZPs from urine by solid-phase extraction with a mixed-mode phase (OASIS® HLB cartridges). Chromatographic separation was performed with a Waters XTerra MS C18 [150 x 2.1 mm (i.d.); bead size, 5 µm] reversed-phase column with deuterated analogs of the analytes as internal standards (IS). Detection was performed with a triple-quadruple mass spectrometer that monitored 2 specific transitions per compound in the electrospray, positive-ion selected-reaction monitoring mode. We tested this technique on urine samples from 12 healthy volunteers and 1 forensic sample obtained in a case of alleged drug-facilitated sexual assault.

Results: Chromatographic separation was achieved within 18 min. The linear dynamic ranges extended from 0.02 or 0.1 µg/L (depending on the drug or metabolite) to 50 µg/L. Extraction recovery (range) was 77%–110%. Limits of detection were ≤0.05 µg/L. No ion suppression was seen except for alprazolam, for which baseline decreased by almost 20%. In the forensic urine sample, the method detected alprazolam (3.5 µg/L) and its characteristic metabolite, {alpha}-hydroxyalprazolam (0.17 µg/L).

Conclusion: This method measured low concentrations of BZPs and BZP-like substances and might be useful for analyses of urine in suspected drug-facilitated sexual assault cases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The benzodiazepines (BZPs)1 and BZP-like substances (e.g., zopiclone and zolpidem) are relatively safe drugs that are frequently prescribed for the treatment of stress, panic disorders, sleep disorders, muscle spasms, alcohol withdrawal, and seizures (1). Because of the high consumption of these substances world-wide, they are involved in many forensic cases. The influence of BZPs on driving and in jobs in which the presence of high serum concentrations in employees could be dangerous has been documented(2)(3)(4)(5)(6). These drugs also play a role in drug-facilitated sexual assault (DFSA)(7)(8).

Zopiclone and zolpidem belong to a new generation of hypnotics and sedatives that are structurally quite different from the BZPs (9). They have a rapid onset of action and a short half-life and, unlike BZPs, have weak myorelaxant and anticonvulsant effects. They are prescribed as hypnotics instead of BZPs(10).

Many widely used screening and confirmation methods do not detect lower concentrations of these drugs, and in some cases, the laboratory may not have the capability to detect a particular drug (11). New ionization techniques have made liquid chromatography–mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS) extremely effective for the analysis of BZPs and BZP-like drugs. We developed an LC electrospray ionization (ESI)-MS/MS method for screening and quantification of zopiclone, zolpidem, and several BZPs and their relevant metabolites, with a focus on the analysis of urine samples from DFSA cases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals, reagents, and solutions
The calibrators used were 1-hydroxymidazolam, 4-hydroxymidazolam, midazolam, 7-aminoclonazepam, desmethylflunitrazepam, flunitrazepam, and 7-aminoflunitrazepam (all from Lipomed); alprazolam, bromazepam, clonazepam, triazolam, lorazepam, 3-hydroxyflunitrazepam, zolpidem, and zopiclone (all from Sigma); flurazepam, desalkylflurazepam, {alpha}-hydroxyalprazolam, {alpha}-hydroxytriazolam, and the deuterated internal standards (IS) 7-aminoflunitrazepam-d7, 7-aminoclonazepam-d4, alprazolam-d5, triazolam-d4, {alpha}-hydroxyalprazolam-d5, desmethylflunitrazepam-d4, {alpha}-hydroxytriazolam-d4, desalkylflurazepam-d4, and flunitrazepam-d7 (all from Ceriliant); lormetazepam (kindly provided by Schering, Levallois-Perret, France); and loprazolam (kindly provided by Sanofi-Aventis, Paris, France).

Propanol-2 (Normapur), dichloromethane (Normapur), and ammonia (Normapur) were purchased from Prolabo. HPLC-grade acetonitrile (Normapur) and methanol (Normapur) were obtained from Carlo-Erba. Formic acid and ammonium formate (>99% pure for both) were purchased from Sigma. Deionized water was prepared on a Direct-Q laboratory plant (Millipore). For sample extraction, Waters Oasis® HLB extraction cartridges (3 mL/60 mg) were used.

We prepared a pH 7.4 Sorensen buffer (66.7 mmol/L) by dissolving 9.07 g of potassium dihydrogen phosphate into 1 L of deionized water and by dissolving 11.6 g of disodium hydrogen phosphate anhydrous into 1 L of deionized water. The disodium hydrogen phosphate solution was then used to adjust the potassium dihydrogen phosphate solution to pH 7.4. A solution of 0.5% ammonia in methanol–water (40:60 by volume) was prepared by adding 1.25 mL of concentrated ammonia to a mixture of 99.5 mL of methanol and 149.25 mL of water.

Stock solutions of desalkylflurazepam, {alpha}-hydroxytriazolam, and {alpha}-hydroxyalprazolam were prepared at 10 mg/L in methanol; all stock solutions of the deuterated standards were prepared at 1 mg/L, and those of the remaining drugs at 1 g/L in methanol. All solutions were kept at –20 °C in the dark. The drugs are stable for at least 3 months under these conditions. Working solutions of mixtures of the analytes at concentrations of 0.0004, 0.001, 0.002, 0.01, 0.04, 0.2, and 1 mg/L were freshly prepared each day by appropriate dilution in methanol–water (50:50 by volume). In the same way, a 0.1 mg/L working solution was prepared from all the IS stock solutions.

sample preparation
We added 1 mL of Sorensen buffer, 50 µL of working IS solution, and 100 µL of the appropriate working solutions of the parent drugs and metabolites to 2 mL of urine to obtain the following calibration concentrations: 0, 0.02, 0.05, 0.1, 0.5, 2, 10, and 50 µg/L. The tubes were then vortex-mixed for 10 s. Solid-phase extraction (SPE) was performed with Oasis HLB cartridges (3 mL/60 mg) previously conditioned with 2 mL of methanol and equilibrated with 2 mL of water. The mixtures of sample and buffer were loaded on the cartridge and slowly passed through the bed without vacuum. The SPE column was rinsed sequentially with 3 mL of deionized water and 3 mL of 0.5% ammonia in methanol–water (40:60 by volume). Reduced pressure was then applied to the column (maximum of –60 kPa) for 15 min to dry the column. The retained drugs were eluted under gravity with 3 mL of dichloromethane–propanol-2 (75:25 by volume) into 5-mL round-bottomed glass tubes. The eluate was evaporated under nitrogen at 40 °C in a Zymark TurboVap® LV Concentration Workstation. The dried extracts were reconstituted in 50 µL of mobile phase, and 15 µL was injected into the chromatographic system.

hplc conditions
The HPLC system consisted of a Shimadzu LC 10 ADvp pump with an SIL-Ht autoinjector and a CTO-10ASvp column oven. The chromatographic separation was performed on a Waters XTerra MS C18 [150 x 2.1 mm (i.d.); bead size, 5 µm] reversed-phase column maintained at 30 °C. The flow rate was 0.2 mL/min. The mobile phase was a gradient of a mixture of acetonitrile and 2 mmol/L (pH 3) ammonium formate (90:10 by volume; solvent A) and 2 mmol/L (pH 3) ammonium formate (solvent B), programmed as follows: initial, 30% A, increased to 36% in 4 min, maintained at 36% for 8.5 min, then increased to 90% A in 3.5 min, and finally, decreased to 30% A in the last 1.5 min. All chromatographic solvents were degassed with helium.

A divert valve was set to direct the LC flow initially to the waste before 1.3 min and after 18 min to protect the spectrometer from salts, polar compounds, and lipids.

ms detection
Detection was carried out with a Thermo-Electron TSQ Quantum Discovery MS/MS system equipped with an orthogonal ESI source and controlled by the Xcalibur computer program. Ionization was achieved with electrospray in the positive ionization mode. The following optimized conditions were used: spray voltage, 4 kV; capillary temperature, 325 °C; sheath gas pressure (N2), 35 kPa; auxiliary gas pressure (N2), 25 kPa.

For selected reaction monitoring (SRM) of the individual compounds and their respective deuterated analogs, the pseudomolecular ions [M+H]+ were selected in the first quadrupole, and the collision energy was adjusted to optimize the signal for the most abundant product ions. A quantification and a confirmation transition were chosen for each compound, except for the deuterated analogs, for which only 1 transition was chosen (Table 1 ). The chromatographic run was divided into 3 segments to limit the number of transitions monitored at a time in MS/MS mode and to ensure sensitivity for quantification purposes. In each segment, the compounds were monitored according to their chromatographic separation (Table 1 ).


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  		Table 1. MS and chromatographic characteristics for tested BZPs.

The total LC-ESI-MS/MS method was optimized to detect 32 substances (including 10 IS).

validation procedures
We performed assay validation according to the guidelines of the US Food and Drug Administration (12). We evaluated selectivity by analyzing urine samples from 10 healthy volunteers who did not take any of the targeted compounds for several days before urine sampling and checked for the absence of the compounds of interest by analyzing the samples with the present technique before using them as blanks. We investigated assay linearity by constructing calibration curves (n = 5) using analysis data from a blank urine sample and 8 urine samples enriched with the analytes at concentrations of 0.02, 0.05, 0.1, 0.5, 2, 10, 25, and 50 µg/L. Stable isotope IS were used to correct for variability in the extraction procedure and the effect of the ion suppression produced by the matrix. When it was not possible to obtain a commercially available deuterated analog for any compound, the deuterated analog of another close compound was selected. We determined the best fit for accuracy for the calibration curves by applying linear and quadratic regression and different types of weighting (1/x, 1/x2).

Extraction recoveries were determined in triplicate at 3 concentrations for all compounds studied: the lower limit of quantification (LLOQ) was 0.02 or 0.05 µg/L, depending on the molecule; the upper limit of quantification (ULOQ) was 50 µg/L; and the intermediate limit was 2 µg/L. In the case of bromazepam and {alpha}-hydroxyalprazolam, the extraction recoveries were studied only at 2 and 50 µg/L because the first concentration (0.02 or 0.05 µg/L) was outside their calibration ranges. For each concentration, 3 urine samples were enriched with the appropriate amounts of drugs and with the IS, and in 3 other samples, only the IS was added. After extraction and evaporation, the first 3 samples were reconstituted with 50 µL of mobile phase and the last 3 samples with 50 µL of mobile phase containing a nominal amount of the compounds of interest. The relative extraction efficiency was calculated as the quotient of the peak-area ratios of the extracted samples with those of the unextracted solutions, which represented 100% recovery.

We evaluated within-run precision and recovery by analyzing 6 samples in the same batch, enriched with all substances studied, for 3 or 4 concentrations of the calibration set, depending on the linear dynamic range of the compound. We evaluated between-run precision and recovery, as well as the linearity of the method, by preparing and analyzing 1 set of calibrators each day for 5 days.

Required specifications for within- and between-batch experiments were a CV <15% (precision) and a mean relative error (MRE) <15% from the nominal value at every concentration studied, except for the LLOQ, where 20% was acceptable for both values (12)(13)(14). The limit of detection was defined as the lowest concentration of the drug giving a signal-to-noise ratio >3:1 and was determined for all compounds by analysis of a blank urine enriched with decreasing amounts of the analytes. The LLOQ was the lowest concentration of the calibration curve that could be measured with an imprecision and deviation from target concentration <20%, in terms of CV and MRE, respectively(12)(13)(14).

We investigated ion suppression by injecting 5 extracts of different blank human urine samples into the system while a mixture of the BZPs in mobile phase was continuously infused in parallel at 50 µL/min in the ionization source through a PEEK tee-junction. We investigated the stability of the extracts in the autosampler by extracting, in quadruplicates, a urine sample enriched with the 21 compounds at 10 µg/L. The second extract was injected 6 h after the first, the third extract 12 h after, and the fourth 15 h after.

applications
This technique was used in a clinical trial approved by our local ethics committee. Twelve healthy volunteers who gave informed consent were administered a single dose of bromazepam, clonazepam, flunitrazepam, lorazepam, zolpidem, or zopiclone (2 patients per drug) at the lowest dosage available. Urine samples were collected every 12 h for 1 week after dosing. The technique was also applied to a forensic sample obtained in a case of alleged DFSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Separation of the 21 compounds and their respective IS was achieved in 18 min. A 6-min equilibration time was necessary at the end of each run. The extracted ion chromatograms of the 21 BZPs obtained for a urine sample enriched at 2 µg/L are presented in Fig. 1 . The selectivity of the method was acceptable in terms of absence of interference in the blank urine samples analyzed. On the total ion chromatogram, not all of the chromatographic peaks resolved, but this was not a problem because quantification and specific identification were performed for each compound with the corresponding transitions in the SRM mode. The combination of retention time (RT) and 2 transitions (and their relative abundances) provided high specificity for all of the compounds. However, with this analytical method, we could not distinguish {alpha}-hydroxytriazolam from 4-hydroxytriazolam because we obtained exactly the same RT and SRM transitions for both, with the same intensity ratio. We therefore introduced {alpha}-hydroxytriazolam, but not 4-hydroxytriazolam, into the calibration and control samples. In addition, 2 peaks showed up on the reconstituted chromatogram of lorazepam: the first peak corresponded to lorazepam and the second to the IS, flunitrazepam-d7 (transition m/z 321.1->246.2). However, these 2 compounds were perfectly separated, so that flunitrazepam-d7 could be kept as IS. The SRM transitions, optimized collision energies, and RT are shown in Table 1Up .


Figure 1
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  		Figure 1. Analysis of blank urine samples enriched with 2 µg/L each of the studied compounds.

TIC, total ion chromatogram.

The deuterated IS selected for each compound is shown in Table 2 . No cross-talk interference with the deuterated IS was observed. The peak-area ratios of the drugs to their respective deuterium-labeled IS were plotted against the corresponding enriched concentrations. The linearity of the method was verified over the concentration range. After assaying different types of regression and weighing factors, we constructed the calibration curves using a 1/x weighted quadratic regression to obtain the best fit across the calibration range, based on the standard error of the fit and minimization of bias of the low calibrators. The squared correlation coefficient (r2), y-intercept, and slope of the regression line are summarized in Table 2 . The mean r2 (n = 5) was >0.998 for all of the compounds except for 4-hydroxymidazolam (r2 = 0.998).


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  		Table 2. Calibration data and limits of detection for the examined compounds.

Limits of detection between 0.01 and 0.02 µg/L were observed for all of the compounds except for bromazepam and {alpha}-hydroxyalprazolam (0.05 µg/L). The LLOQ and ULOQ values for the compounds are shown as the linear dynamic range in Table 2Up . The within-run imprecision (CV) was <17% in all cases for the LLOQ, and was >15% for only 3 drugs: loprazolam, bromazepam, and {alpha}-hydroxytriazolam. For the other concentrations, only one CV was >15% (16.4% for loprazolam at the 2 µg/L concentration). The within-run deviation from target was satisfactory for all compounds under the noted criteria, except for loprazolam, for which the MRE value reached 24% at the LLOQ, and bromazepam, for which it reached 22.4% and 21.2% at the LLOQ and 2 µg/L, respectively.

The between-run CV was <15% for all compounds at all concentrations of the calibration range, including the LLOQ, except for zolpidem, which had a slightly higher result for the LLOQ (CV = 15%), although it was still acceptable according to the Food and Drug Administration recommendations. For all substances, between-run MRE values were ≤14% at all concentrations, including the LLOQ.

The extraction recovery of the analytes at 3 concentrations with the SPE procedure was 79.0%–99.3% at the LLOQ, 77.2%–99.6% at the intermediate concentration, and 88.5%–110.0% at the ULOQ (Table 3 ). No significant ion suppression occurred during chromatographic runs, except for alprazolam, for which the baseline decreased almost 20% (see the Data Supplement that accompanies the online version of this article at http:www.clinchem.org/content/vol52/issue7). The stability of the extracts in the autosampler, studied with an enriched urine sample extracted in quadruplicate (Table 4 ), showed that 4-hydroxymidazolam may begin to deteriorate after 6 h and midazolam and loprazolam after 12 h. This instability of BZPs in solution, despite low temperatures (the autosampler was cooled at 4 °C in this study) and protection from light, is well known and probably unavoidable. As a consequence, when large batches of samples were to be analyzed, a portion of each extract was stored dry at 4 °C before being reconstituted and placed in the autosampler a few hours before analysis.


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  		Table 3. Extraction recovery of the 21 BZPs and metabolites.


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  		Table 4. Stability in the autosampler of extracts of the 21 BZPs and metabolites at 10 µg/L.

The clinical trial in healthy volunteers given a single dose of bromazepam, clonazepam, flunitrazepam, lorazepam, zolpidem, or zopiclone (2 patients per drug) at the lowest dosage available showed that lorazepam, bromazepam, and 7-aminoflunitrazepam could be detected in urine up to 144 h (1 week) post dose, whereas flunitrazepam was not detected after 132 h, zopiclone after 84 h, and zolpidem after 48 h in one volunteer and after 96 h in the other. These results demonstrate the suitability of this method, which we have used successfully in our laboratory for the investigation of actual DFSA cases.

The results obtained in a DFSA case are shown in Fig. 2 . In this urine sample, the assay detected alprazolam at 3.5 µg/L and its characteristic metabolite, {alpha}-hydroxyalprazolam, at a low concentration of 0.17 µg/L.


Figure 2
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  		Figure 2. Chromatograms of a forensic DFSA sample collected <24 h after the assault.

The urine sample contained alprazolam and its metabolite {alpha}-hydroxyalprazolam at 3.5 and 0.17 µg/L, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because higher concentrations of drugs and their metabolites can usually be found in urine, it is the specimen of choice for toxicologic testing in DFSA cases (15). One of the most important issues in DFSA investigations is the sensitivity of the screening and confirmatory techniques because some of the compounds (e.g., BZPs) are typically used in a single low dose(16)(17). Kintz et al.(18) demonstrated that the LC-MS and gas chromatography (GC)-negative chemical ionization-MS/MS techniques are the most sensitive for detecting low quantities of the drug metabolite 7-aminoflunitrazepam several days after administration of flunitrazepam. A highly sensitive GC-negative chemical ionization-MS method for the simultaneous quantification of clonazepam and its major metabolite in urine detected the metabolite 28 days after a single dose (3 mg) of clonazepam(19). Other LC-MS (and LC-MS/MS) assays have been reported for forensic applications, including use in DFSA cases(19)(20)(21)(22)(23).

We evaluated several previous procedures for extraction of the BZPs and related compounds during development of the method we report here (24). The presence of compounds in a relatively wide polarity range was the principal reason for our use of an SPE method. Variation in the pH of urine samples induced significant variations in compound retention on the SPE column and in chromatogram background noise. The best results were obtained by dilution of urine samples with Sorensen buffer (pH 7.4). Because of the high sensitivity of our method, which allowed detection of the parent drug and phase I metabolite at very low concentrations, and the instability of several analytes during incubation (mainly the amino metabolites), we processed the urine samples without enzymatic hydrolysis.

Our results demonstrate that this technique meets method validation criteria for measuring analytes of interest in urine. In the case of bromazepam and loprazolam, certain values were not in the expected range, but this should not present a problem for applying this technique to urine samples in DFSA cases, where precise quantification is not the most important goal.

The use of HPLC to separate the compounds before detection by MS avoids the temperature-induced degradation of certain BZPs that can occur when GC methods are used (10). Another major advantage of HPLC for analyzing BZPs is that no derivatization of the metabolites is required(11).

Compared with other reports on analysis of several BZPs (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), in the screening method described in this study, the LLOQs for 19 BZPs or their major metabolites and 2 related compounds (zopliclone and zolpidem) are lower and seem to be appropriate for the detection and quantification of these compounds in urine samples from DFSA cases.


   Acknowledgments
 
We thank Bea López and Anna Chasnoff for helpful assistance in the preparation of this manuscript. This work was supported by a grant from the Ministry of Education and Science of Spain under the Formación de Profesorado Universitario Program.


   Footnotes
 
1 Nonstandard abbreviations: BZP, benzodiazepine; DFSA, drug-facilitated sexual assault; LC-MS, liquid chromatography–mass spectrometry; MS/MS, tandem MS; ESI electrospray ionozation; IS, internal standard; SPE, solid-phase extraction; SRM, single reaction monitoring; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; MRE, mean relative error; RT, retention time; and GC, gas chromatography.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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