Journalof AnalyticalToxicology,Vol. 30, January/February2006
Determination of Benzodiazepinesin Human Urine usingSolid-PhaseExtractionand High-Performance Liquid Chromatography-ElectrosprayIonization Tandem Mass Spectrometry S. Hegstad*, E. I.. Oiestad, U. Johansen, and A.S. Christophersen Norwegian Instituteof Public Health, Division of Forensic Toxicologyand Drug Abuse, P.O. Box 4404 Nydalen, N0-0403 Oslo
Abstract A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of benzodiazepines, on the market in Norway, and/or their metabolites in human urine. The following compounds were included: 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, ~hydroxyalprazolam, oxazepam, 3-OH-diazepam, and n-desmethyldiazepam. The method includes hydrolysis of urine samples (0.5 mL) with ~-glucuronidase at 60~ for 2 h before solid-phase extraction with a polymer-based mixed.mode column. The analytes were quantified in multiple reaction monitoring mode using two transitions. Deuterated analogueswere used as internal standards for all analytes except 7-aminonitrazepam and ~hydroxyalprazolam, which were quantified using 7-aminoclonazepam-d4and alprazolam-ds, respectively. The concentration range was 0.1-8.0pM for 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, and ~hydroxyalprazolam and 0.5-40pM for the other compounds. The average recovery for the different analytes ranged from 56% to 83%. The between-day precision of the method was in the range of 3-12%. The limits of quantification were found to be between 0.002 and 0.01pM for the different compounds. Comparison with other analytical methods was performed for method validation, using approximately 500 samples provided by the routine laboratory at the Norwegian Institute of Public Health. The LC-MS-MS method has proven to be robust and specific for the determination of benzodiazepines in urine. It has been routinely used for approximately 1800 samples in the past 7 months.
Introduction Benzodiazepines (BZDs) belong to a group of drugs known for their sedative, hypnotic, and anticonvulsant properties and are among the most frequently prescribed drugs in the world for the therapy of anxiety and sleeping disorders (1-3). These 9Author to whom correspondence should be addressed. E-mail:
[email protected].
compounds are, however,also associated with misuse and have become a serious problem in many countries (4). In Norway, BZDs are some of the most frequently detected drugs in blood samples from suspected drugged drivers (5,6). The Norwegian Institute of Public Health receives about 25,000 urine samples each year from prison and probation services, social services, and workplace testing programs. Statistics based on the 5124 urine samples that tested positive after screening with immunological methods show that 50% of the samples contained benzodiazepines. Thus, a robust and specific quantitative method is needed. In urine, BZDs are predominantly excreted as glucuronide conjugates. Flunitrazepam, clonazepam, and nitrazepam are metabolized to 7-amino metabolites by reduction of the 7nitro group or by demethylation to n-desmethyl metabolites (7-9). The 7-amino metabolites are subsequently converted to N-glucuronides, and the n-desmethyl metabolites are further hydroxylated and glucuronidated. Diazepam is converted to 3-OH-diazepam, n-desmethyldiazepam, and oxazepam, which are subsequently conjugated to glucuronides. Alprazolam is metabolized to oc-hydroxyalprazolam and 4-OH-alprazolam (10), but about 10% is excreted in the urine as the parent drug. A number of studies have been reported on the analysis of BZDs and their metabolites in urine. Most of them used gas chromatography-mass spectrometry (GC-MS) techniques, which requires tedious derivatization (11-18). Liquid chromatography (LC)-MS is increasingly being used in forensic toxicologyto quantify a wide range of compounds in biological samples (4,19-25). Most of the studies mentioned used atmospheric pressure chemical ionization (APCI),but electrospray ionization (ESI) has also been used for the determination of BZDs in serum and urine (26-28). LC-MS-MSprovides greater sensitivity and selectivity than LC-MS and has recently been used for determination of BZDs in plasma (29), hair (30,31), larvae (32), and whole blood (33). The purpose of the present study was to developa robust and specific LC-MS-MS method for the simultaneous quantification of BZDs and their metabolites in urine that is suitable for
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31
Journal of Analytical Toxicology, Vol. 30, January/February 2006
routine analysis. The eight compounds included were: 7aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, alprazolam, (z-hydroxyalprazolam,oxazepam,3-OHdiazepam, and n-desmethyldiazepam. Such a method would increase the efficiency of our routine laboratory by replacing two chromatographic methods involving derivatization techniques [high-performance liquid chromatography (HPLC)-fluorescence and GC-MS].
Material and methods
Chemicals and reagents Reference compounds were obtained from four pharmaceutical companies: 7-aminonitrazepam, 7-aminoflunitrazepam, o~-hydroxyalprazolam, and 3-OH-diazepam (Cerilliant Corp. Austin, TX); 7-aminonitrazepam, 7-aminoclonazepam, 7aminoflunitrazepam, alprazolam, and c~-hydroxyalprazolam (Lipomed, Arlsheim, Switzerland); alprazolam, 3-OH-diazepam, and oxazepam (Sigma-Aldrich, St. Louis, MO); oxazepam and n-desmethyldiazepam (Alltech,State College, PA). The internal standards 7-aminoclonazepam-d4, 7-aminoflunitrazepam-dT, alprazolam-ds, oxazepam-d5, 3-OH-diazepam-ds, and n-desmethyldiazepam-d5 were all purchased from Cerilliant Corp. (Austin, TX). The enzyme 13-glucuronidase(TypeLII, from Patella vulgata 1,000,000-3,000,000 units/g solid) was obtained from Sigma-Aldrich. Other chemicals were of HPLC or analytical grade from various commercial sources. The Oasis MCX (60 mg, 3 mL) extraction columns were purchased from Waters (Milford, MA). Standard solutions
Benzodiazepine stock solutions (?-amino metabolites/alprazolam/c~-hydroxyalprazolam,50 IJM and oxazepam/3-OHdiazepam/n-desmethyldiazepam, 2501JM) and deuterated internal standards (30!JM)were dissolved in methanol. Standard and control solutions were prepared by appropriate dilution of a stock solution with water. Standard calibration solutions were prepared in blank urine in concentrations ranging from 0.1 to 8tim (7-amino metabolites, ot-hydroxyalprazolam, and alprazolam) or 0.5 to 401~M(oxazepam, 3OH-diazepam, and n-desmethyldiazepam). Quality control (QC) samples were prepared in blank urine at concentrations of 0.12-0.631aM and 1-51JM, respectively. The internal standard solution was diluted with water to concentrations in the range of 2-6t~M.
column was washed with water (2 mL), 0.1M hydrochloric acid (1 mL), and methanol (2 mL). The analytes were eluted with 2 mL dichloromethane/isopropanol/ammoniumhydroxide (80:20:2, v/v). The eluates were evaporated to dryness under N2 (40~ and dissolved in 200 I~Lacetonitrile/water (1:1, v/v). Instruments
HPLC. LC was performed using a Waters Alliance 2695 system (Manchester, U.K.). Separation was performed on a Waters Symmetry C18 (2.1 x 100 ram, 3.5 ]Jm) column using gradient elution at a flow rate of 0.3 mL/min with the following solvent system: 100% acetonitrile (A) and 5ram ammonium acetate, pH 5 (B) (Table I). The pre-column volume was set to 0.95 mL and the column temperature was held at Table I. Gradient Table* Time (min)
A (%)
B (%)
0.0 5.0 9.0 9.1
10 30 80 10
90 70 20 90
* A linear curve profile for the change in mobile phase composition was used.
Table II. MRM Transitions and Conditions for Each Analyte and Internal Standard
Analyte
Transition (m/z)
Cone Collision Voltage Energy (V) (eV)
7-Aminonitrazepam
251.9 > 121.0 251.9 > 146.0
40 40
23 25
7-Aminoclonazepam
286.0 > 221.9 286.0 > 250.0
40 40
22 18
7-Aminoclonazepam-d4
290.0 > 254.0
40
18
7-Aminoflunitrazepam
283.9 > 135.0 283.9>227.0
50 50
23 23
7-Aminoflunitrazepam-d7
291.1 > 138.0
50
23
a-Hydroxyalprazolam
324.8 > 215.9 324.8 > 296.9
40 60
35 25
Alprazolam
309.0 > 205.0 309.0 > 280.9
40 40
38 23
Alprazolam-ds
314.0 > 285.9
40
23
Oxazepam
286.9 > 162.9 286.9 > 240.9
40 45
35 30
Oxazepam-d5
291.9 > 245.9
45
20
3-OH-Diazepam
300.9 > 176.9 300.9 > 255.0
40 42
35 40
3-OH-Diazepam-ds
305.9 > 260.0
42
16
Extractionprocedure
N-Desmethyldiazepam
Urine samples were diluted with 0.5 mL 0.1M phosphate buffer (pH 7.5) and extracted using an Oasis MCX column, preconditioned with methanol (2 mL) and water (2 mL). The
270.9 >139.9 270.9 > 164.9
50 40
25 25
N-Desmethyldiazepam-ds 275.9> 139.9
50
25
Sample pretreatment Urine samples (0.5 mL) mixed with 50 IJL internal standard and 0.5 mL 0.1M acetate buffer (pH 4.0) were incubated with 13-glucuronidase(approximately4500 units) at 60~ for 2 h for hydrolysis of conjugates.
32
Journal of Analytical Toxicology, Vol. 30, January/February2006
months at-20~ The samples were stored at 4~ for about one 35~ during analysis. The injection volume was 10 I~L. week prior to storage at-20~ A liquid-liquid extraction was MS-MS. A Quattro Ultima Pt tandem-quadropole MS (Waperformed, and the extracts were subsequently analyzed using ters) equipped with a Z-spray electrospray interface was used. the original HPLC-fluorescence method. Briefly, 1 mL urine Positive ionization was performed in the multiple reaction was diluted with 0.5 mL borate buffer (pH 11.1) and subsemonitoring (MRM) mode. The capillary voltage was set to 1.0 quently extracted with ethylacetate/heptane (4:1), before kV; the source block temperature was 120~ and the desolvaderivatization with fluorescamine. tion gas (nitrogen) was heated to 400~ and delivered at a flow rate of 500 L/h. The cone gas (nitrogen) was set to 50 L/h, and the collision gas (argon) pressure was maintained at 0.5 Specificity psi. The MRM transitions, the cone voltage, and the collision In order to investigate the specificity of the method, samples energy for the measurement of the benzodiazepines and the incontaining high concentrations of drugs such as amternal standards are shown in Table II. Two transition ions phetamines, ecstasy, opiates, cocaine, cannabinoids, zopiclone, were monitored for the analytes and one for the internal stanzolpidem, and methadone were analyzed. The sample pretreatment, extraction procedure, and quantification were perdards. System operation and data acquisition were controlled using Mass Lynx4.0 software (Waters). All data were processed with the QuanLynx quantification pro7-Aalinoclonazcpam 288 > 250 gram (Waters) loo
too.+
Method validation The four-point calibration curves were based on peak-height measurements versus peak-height of the corresponding internal standard. The extraction recovery for the solid-phase extraction (SPE) method was determined at two concentration levels (QC samples) with six replicates at each level. Recovery was estimated by comparison of the peak heights obtained when the analytes were added before sample preparation with those obtained when the analytes were added after the extraction step. In both cases, the internal standards were added after the extraction step. Within-day precision was estimated by analysis of 10 separate preparations of each QC concentration level in a single assay. Between-day precision was determined by analysis of preparations of each QC concentration on 10 different days over a 2-month period. Drug-free urine supplemented with various concentrations of analytes (0.1-0.0021~M)was extracted and analyzed in six replicates to determine the limit of quantification (LOQ). The results were compared with those from routine analysis of approximately 500 samples. A GC-MS method (34) was used for oxazepam, 3-OH-diazepam, and n-desmethyldiazepam. The 7-amino metabolites were originally analyzed by a modified HPLC-fluorescence method (35). 7-Amino metabolites with or without enzymatic hydrolysis In order to determine the free 7-amino metabolites, two parallels of 25 authentic urine samples, one including pretreatment with enzyme as described previously and one without this step, were analyzed using the new LC-MS-MS method.
Stability of 7-amino metabolites Stability was tested by analyzing authentic urine samples (n = 25) before and after storage for I to 8
7-Aminotlunitrazepam
%Z1~. . . . . .~. +. + . . .Z+a , . . . . . .~.PS . . . . . . . . ++O ...
~o]
.~
- . . . ++~~. . . . . a. .. 7 .+. . . ~~++ . . . . . . 2~1
s
:::2
,.o..o+,o
t ...... ,
N'Desmethyl~azr
l
loo,
++l
271 >
140
Time (min)
Figure 2. MRM chromatograms of analytes of an authentic sample. Determined concentrations: 7-aminoclonazepam (0.15pM), 7-aminoflunitrazepam (0.67pM), alprazolam (0.07I~M),c~-hydroxyalprazolam(0.41pM), oxazepam (0.49pM), 3-OH-diazepam (0.87[aM), and n-desmethyldiazepam (0.63tJM).
33
Journal of AnalyticalToxicology,Vol. 30, January/February2006
formed as described for the LC-MS-MS method.
Results and Discussion
Ion suppression (matrix effects) For the investigation of ion suppression, a stock solution containing all eight benzodiazepines was prepared in acetonitrile/water (50:50), which was continuously infused postcolumn into the MS-MS by a syringe pump. Aliquots of 10 ~L of blank extracted urine and authentic samples containing various drugs were injected into the HPLC (36,37).
An initial survey showed that the 7-amino metabolites, 7aminonitrazepam in particular, were difficult to detect with LC-MS because of coeluting compounds. To achieve high specificity and signal-to-noise ratio, LC-MS-MS detection was therefore required. During method development, direct injection into the chromatographic system after dilution of the hydrolyzed urine samples was evaluated. This sample pretreatment method worked well for the diazepam metabolites, but not for the other compounds. An extraction method was therefore required for quantitative determination of 7amino metabolites, alprazolam, and ~-hydroxyalprazolam. APCI is known (4,29) to assure efficient ionization of benzodiazepines (e.g., 7-aminoflunitrazepam). However, because the sensitivity obtained using our instrumentation was at
Identification and quantification The analyte specimens were identified by comparing their retention times with the retention times of the corresponding standards and control samples. The ratio between the two MRM transitions for each analyte was also compared with those of the corresponding standards and control samples and should not deviate more than 5% from the average ratio. Peak height was used for all quantification. 7-Aminoclonazepam-d4 and alprazolam-d5were used as the internal standard for quantification of 7-aminonitrazepam and (~-hydroxyalprazolam.
Table III. Concentration Range of Calibration Curves Concentration Analyte
Statistical analysis Passing-Bablok regression (38) was used to investigate agreement between the original GC-MS and the new LC-MS-MS methods using "Analyse-it" for Microsoft Excel vl.72 (Analyse-it Software, Ltd., Leeds, U.K.). The PassingBablok method is useful when there is imprecision in both variables (X and Y). Imprecision need not be normally distributed and may have nonconstant variance over the sampiing range. Furthermore, the regression line is not unduly biased by extreme values.
(pM)
7-Arninonitrazepam 7-Aminoclonazepam 7-Aminoflunitrazepam ~-Hydroxyalprazolam Alprazolam Oxazepam 3-OH-Diazepam N-Desmethyldiazepam
0.1-8 0.1-8 0.1-8 0.1-8 0.1-8 0.5-40 0.5-40 0.5-40
Table IV. Between-Day Precision and Accuracy (n = 10), Recoveries and LOQ of the Analytes
Recovery
Mean
Concentration Found(pM)
RSD
Accuracy
(%)
RSD
LOQ
(%)
(%)
(n = 6)
(%)
(pM, n = 6)
0.126
0.113
10.7
-10.3
56
3.8
0.01
1.0]
0.91
6.7
-9.9
67
2.9
7-Aminoclonazepam
0.124 0.99
0.118 0.94
10.0 6.8
-4.8 -4.9
66 76
4.5 7.9
0.01
7-Aminoflunitrazepam
0.125 1.00
0.131 1.05
9.5 3.2
4.5 4.5
73 80
6.1 5.6
0.002
o~-Hydroxyalprazolam
0.125 1,00
0.123 1.05
11.6 7.3
-1.6 5.2
83 85
5.2 7.5
0,002
Alprazolam
0.125 1.00
0.120 0.97
6.1 8.4
-3.8 -3.3
83 89
3.1 3.9
0.002
Oxazepam
0.623 4.98
0.620 4.74
4.6 4.8
-0.7 -4.7
70 65
3.1 4.6
0.01
3-OH-Diazepam
0,625 5.00
0.700 5.33
7.9 4.7
11.4 6.5
57 51
5.0 4.0
0.01
N-Desmethy[diazepam
0,623 4.99
0.610 4.88
6.7 7.3
-2,8 -2.2
68 74
2.3 6.1
0,01
Analyte 7-Aminonitrazepam
34
Added (pM)
Journal of Analytical Toxicology, Vol. 30, January/February2006
7-Aminometabolites
least 10 times higher using ESI than using APCI, ESI was preferred. Figure 1 and 2 present MRM chromatograms of the lowest-concentration standard and an authentic urine specimen, respectively.
7-hminonitrazepam, 7-aminoclonazepam, and 7-aminoflunitrazepam were previously analyzed by an HPLC-fluorescence method that only determined the free 7-amino metabolite concentrations (no enzymatic hydrolysis) and that was not directly comparable with the new method (which includes enzymatic hydrolysis). Enzymatic hydrolysis increased the concentration of 7-amino metabolites by between 10% and 80% (data not shown). Comparison of hydrolyzed samples run by both methods showed good agreement for samples run at the same time. However, after storage for some time at -20~ in the freezer, the concentration decreased regardless of the method used. If the same sample was analyzed at long intervals, different results were obtained even if the same method was used (HPLC-fluorescence method). The level of 7-amino metabolites decreased by approximately 10% to 100% when samples were stored in the freezer. No correlation was found between storage time and the decrease in concentration (data not shown). This discrepancy is probably because of the instability of the analytes in urine. 7-Amino metabolites have previously been reported to be unstable in postmortem blood (39). Alprazolam and c~-hydroxyalprazolamhave not previously been analyzed routinely in urine specimens at the Norwegian Institute of Public Health, but analysis of 5 samples provided by another laboratory showed good correspondence (40).
Method validation The increased sensitivity obtained using SPE resulted in nonlinear calibration curves for oxazepam and 3-OH-diazepam. In order to obtain approximately linear calibration curves, the collision energy of oxazepam and 3-OH-diazepam was increased, decreasing the analyte signal. Calibration curves were made for each compound in the concentration range listed in Table III. A weighted (l/x) second order regression line was applied for each compound, and the calibration curves were found to be reproducible in the concentration range listed. The between-day precision, accuracy, extraction recoveries, and LOQ of the analytes are presented in Table IV. The relative standard deviations (RSDs) ranged between 3.2% and 11.6%. The within-day variation was better, with RSDs between 1.8% and 4.5% (data not shown). The extraction recoveries of the analytes at the lowest concentration level were above 56%, with RSDs up to 8%. The lowest calibration standard for all analytes was set at least 10 times higher than the LOQ of the method because this cut-off was considered suitable for confirmation of screening positive specimens.
Comparisonof the new LC-MS-MSmethodwiththe previousmethodfor oxazepam,3-OH-diazepam,and n-desmethyldiazepam
Specificity To investigate the specificity of the method, benzodiazepinenegative specimens containing high concentrations of drugs such as amphetamines, ecstasy, opiates, cocaine, cannabinoids, zopiclone, zolpidem, and methadone were analyzed. No falsepositive results were detected, and no false-positive samples were detected during the validation period, when approximately 500 benzodiazepine-positive EMIT| specimens were analyzed. However, another compound giving rise to peaks for both transitions of 7-aminonitrazepam with an acceptable ratio has been discovered with increasing frequency in routine use. Differentiation was possible based on a difference in retention time of approximately 0.1 min. It has been identified as a metabolite of mirtazapine (an antidepressant) (41).
Approximately 500 samples were analyzed using both the new LC-MS-MS method and the routine GC-MS method. Passing and Bablok regression analysis (Figure 3) showed a linear relationship between the two methods. The methods were compared in the concentration range 0.5-5pM, the latter being the highest concentration used for the standards in the GC-MS method. 3-OH-diazepam and n-desmethyldiazepam showed a good correlation between the two methods, whereas the new method gave slightly lower concentrations for oxazepam. However, the difference was acceptable considering the purpose of the method. 69
Identity line
4-
B
, ~
ldRnt~ line
/
5 4.5 4.
4-
,~;s
~2,5
J
1-
eJ
~ -
0
o
;
~
"
1.5.
1 y=O.g571x+O.0286=. .
0
ro
2.5. ,,- ~
Identity tine
~
GC-MS (laM)
~
~
0.5 0
y,== 1.00g4x-_ 0.058
F
0.5. 0
;
i GC-MS (pM)
i
4
1
2
3
4
5
GC-MS (p.M)
Figure 3. Passing-Bablok regressionanalysisfor the assessmentof agreement between the LC-M~MS method presented here and the reference GC-MS method after analysisof oxazepam (n = 128) (A), 3-OH-diazepam (n = 76) (8), and n-desmethyl-diazepam (n = 67) (C).
35
Journal of Analytical Toxicology,Vol. 30, January/February2006
Ion suppression (matrix effect) Benzodiazepine-negative urine specimens were spiked with a low QC sample to investigate whether other compounds in the sample affected the analytical signal of the analytes. An effect was seen for some specimens, but no correlation with the other known substances in the samples could be seen, and this was therefore evaluated to be a possible matrix effect. For methadone, a suppressing effect on the peak height of alprazolam/alprazolam-d5 could be seen. This has an impact on the result for r which uses alprazolam-d5 as the internal standard. Special care should therefore be taken in evaluating the result for ~-hydroxyalprazolam if aiprazolam-ds has an unusually low signal. No ion-suppressant effect could be seen for the partly coeluting compounds 3-OH-diazepam and n-desmethyldiazepam in a comparison with the original GC-MS method. An effect of coeluting matrix compounds in ESI has previously been described (36,37,42,43), and ion suppression tends to decrease the analytical signal. The possibility of a matrix effect was evaluated by the experimental technique developed by Bonfiglio et al. (42). The post-column infusion experiment did not show any matrix suppression for injection of blank pretreated matrix or authentic specimens containing various drugs. However, a variation of the internal standard signal was observed during method development. Though it is necessary for adequate sensitivity and general sample clean-up, SPE can lead to increased concentrations of interfering substances (43). The internal standard signal tends to decrease as the concentration of analyte increases but can be seen to decrease or increase for low or zero concentrations of compounds as well. Our strategy has therefore been to use deuterated analogues as internal standards to try to compensate for the observed ion suppression effect. Special care is taken in evaluating specimens containing 7-aminonitrazepam and (z-hydroxyalprazolam, which do not have their own deuterated internaI standards, and specimens with analyte concentrations close to cut-off, which might become false positives.
Conclusions A robust and specific quantitative method for determination of eight benzodiazepines in urine combining mixed mode SPE and LC-MS-MS has been developed. It has been validated with satisfactory results in regards to separation, recovery,linearity, and specificity. [t has been routinely used for approximately 1800 samples in the past 7 months and has significantly improved the efficiencyof routine analysis by replacing two chromatographic methods, which involved time-consuming derivatization techniques (HPLC-fluorescence and GC-MS).
Acknowledgments The authors would like to thank Anne Smith-Kielland and Jorg Morland for critical reading of the manuscript.
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