Analysis of Benzodiazepines in Blood by LC/MS/MS Application Note

Forensic Toxicology

Authors Introduction Hazel Rivera and G. Stewart Walker The analysis of benzodiazepines is of great interest Flinders University to forensic toxicologists. Adelaide, South Australia Australia Screening of these compounds has been problem- atic since immunoassays are often not sufficiently Peter Stockham and D. Noel Sims specific or sensitive enough for low-dosage benzo- Forensic Science South Australia diazepines, especially in blood. Benzodiazepines Adelaide, South Australia have been analyzed using gas chromatography/ Australia nitrogen phosphorus detector (GC/NPD) [1], gas John M. Hughes chromatography/electron capture detector (GC/ECD) [1], and gas chromatography/mass spec- Agilent Technologies, Inc. trometry (GC/MS) [2, 3]. Many benzodiazepines are Pleasanton, CA polar and thermally labile, making them diffi-cult, USA if not impossible, to analyze with GC or GC/MS without derivatization. Some of the com- Abstract pounds cannot be derivatized for improved chromatographic behavior. A sensitive and selective method for the simultaneous screening and identification of 13 benzodiazepines and Screening for benzodiazepines can also be carried 5 metabolites in human blood using the Agilent LC/MSD out using HPLC with UV detection [4], but this Trap is described. The method uses liquid-liquid extrac- technique lacks both the sensitivity and specificity tion followed by reverse-phase LC/MS/MS (liquid required for forensic applications. Furthermore, chromatography/tandem mass spectrometry). The some of the newer benzodiazepines, like fluni- technique is suitable for screening analysis and trazepam, have much lower usage dose ranges and high-confidence identification of the analytes at their faster clearance, and therefore require identifica- lowest reported dosage concentrations using only 500 µL tion at lower levels. of blood and the original model ("Classic") of the Agilent LC/MSD Trap. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines. Liquid chromatography/mass spectrometry Experimental (LC/MS) is ideally suited for this family of compounds because the technique does not require Sample Preparation derivatization, thereby saving time, expense, and experimental difficulty. This class of compounds Reference solutions of each analyte were combined, also ionizes well in either electrospray ionization diluted in water and added to drug-free blood, (ESI) or atmospheric pressure chemical ionization along with the internal standard, to prepare cali- (APCI) modes, and therefore can be easily detected brators at low, medium and high dose con- at µg/mL levels and below. A large number of ben- centrations of each drug. Typical low and high zodiazepines and related substances have also been concentrations are shown in Table 2. The analyzed using the Agilent single quadrupole liquid extraction method is the same as used for chromatography/mass selective detector screening of these drugs by GC/ECD and GC/MS, (LC/MSD) in APCI mode and selected ion monitor- with any derivatization step omitted and the final ing (SIM) mode [5]. The full scan sensitivity of the residue dissolved in the initial mobile phase rather Agilent LC/MSD Trap allows for both identifica- than in a typical GC solvent. tion/confirmation and quantitation in a single analysis, and the multiple reaction monitoring Table 2. Concentration Ranges for Analytes (mg/L or µg/mL) (MRM) mode provides for more specific detection Benzodiazepine/ Low High in a complex matrix such as blood. metabolite dosage dosage In this work 13 benzodiazepines and 5 metabolites Alprazolam 0.01 0.1 Bromazepam 0.08 0.2 [Table 1] are analyzed in a single run of approxi- Clobazam 0.1 1 mately 20 minutes. This application note is derived Clonazepam 0.03 0.08 from work carried out in the Australian laboratory 7-aminoclonazepam 0.03 0.14 and previously published in reference 6. Diazepam 0.05 2 Nordiazepam 0.05 2 Flunitrazepam 0.005 0.02 Table 1. Compounds Analyzed 7-aminoflunitrazepam 0.002 0.02 Flurazepam 0.0005 0.028 Benzodiazepines Metabolites N-desalkylflurazepam 0.04 0.15 Alprazolam – Lorazepam 0.02 0.3 Bromazepam – Midazolam 0.08 0.25 Clobazam – Nitrazepam 0.03 0.2 Clonazepam 7-aminoclonazepam 7-aminonitrazepam 0.03 0.2 Oxazepam 0.15 2 Diazepam Nordiazepam Temazepam 0.3 1 Flunitrazepam 7-aminoflunitrazepam Triazolam 0.002 0.02 Flurazepam N-desalkylflurazepam Lorazepam – To 0.5-mL blood in a glass screw-top tube was Midazolam – added 50 µL of freshly prepared internal standard Nitrazepam 7-aminonitrazepam working solution (5 µg/mL in water). To this tube Oxazepam – was added 1.75 mL of 4.5% ammonia solution and Temazepam – 10 mL of 1-chlorobutane, and the contents rolled Triazolam – on a mechanical mixer for 10 minutes. After cen- trifuging, the solvent was drawn off, transferred to Prazepam (internal standard) a clean glass tube and evaporated to dryness in a Jouan centrifugal evaporator. The residue was dissolved in 100 µL of mobile phase.

LC/MS/MS Instrumentation

The LC/MS/MS system used in this work consisted of an Agilent 1100-series vacuum degasser, binary pump, autosampler, thermostatted column com- partment, diode array detector (DAD) with micro- flow cell, an LC/MSD Trap “Classic” (model

2 G2445A, equivalent in performance to the current separation in a reasonable time. Because APCI has VL model), and a G1947A APCI source. Complete also been demonstrated to be less susceptible to system control and data analysis was provided by matrix suppression effects than ESI, and because the Agilent LC/MS ChemStation. both flurazepam and lorazepam showed better sensitivity with APCI, it was chosen as the preferred ionization method.

LC/MS Method Details Various mobile phase compositions were evalu- LC Conditions ated, with the objective being a best compromise Column: Agilent ZORBAX Eclipse XDB-C8, among a simple LC method, reasonably short run 150 × 4.6 mm, 5 µm (p/n 993967-906) time, and maximizing chromatographic resolution Mobile phase: A = 20 mM ammonium formate, of the analytes. The choices included isocratic and pH 9 in water gradient methods using either 20-mM ammonium B = methanol formate at pH 3 or pH 9, or 0.1% formic acid. Once Flow rate: 0.7 mL/min APCI was chosen as the preferred ionization method, methanol became the organic component Gradient: 60% B until 15 min of choice over acetonitrile, as it provides better 100% B at 16 min 100% B to 21 min sensitivity in APCI and does not build up carbon Post-time (column re-equil): 5 minutes deposits on the APCI corona needle. Acetonitrile (sufficient for reproducible retention times) has higher gas phase basicity than methanol; since Injection vol: 5 µL ionization in APCI occurs in the gas phase (rather in the liquid phase as in ESI), acetonitrile can com- MS Conditions pete with analyte molecules for the available Source: Positive APCI protonation “work”. Nebulizer: 60 psig The basic aqueous phase with methanol as the Vaporizer: 400 °C organic component was found to give the best sep- Drying gas flow: 5 L/min aration, chromatographic peak shape, and sensitiv- Drying gas temp: 350 °C ity for this analysis. The ZORBAX Eclipse C8

Vcap: 3000 V column is stable at the effective pH of this mobile Corona: 4 µA phase for extended periods of time. The C8 station- ary phase proved to be sufficiently retentive even Scan: m/z 150–400 for the polar metabolites; C18 would have required Averages: 2 longer run times. SPS settings: Target mass m/z 300 Reconstituting the sample extracts in the initial Compound stability 60% (Skim 1: 24 V, mobile phase, a recommended practice for HPLC, Cap exit offset: 69 V) was found to give better peak shapes and therefore Trap drive 100% (resulting value 27) better sensitivity than using simply methanol/ Precursor isolation 2.5 amu water. This is especially important for the width: early-eluting polar analytes. Cutoff: 45% (113–175 m/z for these compounds) Prazepam was chosen as a suitable internal stan- MRM: Eight time segments as shown in Table 3 dard because of its structural similarity to the other analytes and because it is not prescribed in Results and Discussion Australia.

Discussion The optimum fragmentation amplitude for each analyte was determined by infusing a 5-µg/mL Both ESI and APCI were evaluated for the analysis solution of a single compound into the MS/MS, and of these compounds. Generally, both gave good increasing the fragmentation amplitude until the sensitivity at the low dosage levels needed for the precursor ion intensity was reduced to 10%–20% of method. However, both flurazepam and lorazepam its major product ion response. The resulting value showed poor response in ESI with the mobile was used in the data acquisition method as shown phase which gave the best chromatographic in Table 3.

3 Table 3. Data Acquisition Parameters for MRM Precursor Major Fragmentation Fragmentation Group Benzodiazepine/ RT ion Product ionamplitude width number [min] metabolite (min) [M+H]+ (m/z)(V)(m/z) 1 7-aminonitrazepam 2.6 252 224 2.00 10 [1.00–4.00] 7-aminoclonazepam 2.8 286 250 2.50 10 7-aminoflunitrazepam 3.1 284 264 1.88 10 2 Bromazepam 5.3 316 288 1.92 10 [4.00–5.70] 3 Clonazepam 6.1 316 270 2.00 10 [5.70–6.70] Nitrazepam 6.2 282 236 1.86 10 Flunitrazepam 6.3 314 268 1.90 10 4 Clobazam 7.3 301 259 3.37 40 [6.70–8.80] Flurazepam 7.6 388 315 2.60 40 Triazolam 7.8 343 308 3.57 40 Alprazolam 8.3 309 281 4.67 40 Lorazepam 8.3 321 275 2.98 40 Oxazepam 8.6 287 241 3.32 40 5 N-desalkylflurazepam 9.2 289 261 4.57 40 [8.80–11.10] Temazepam 9.6 301 255 3.72 40 6 Nordiazepam 12.1 271 243 1.88 10 [11.10–13.00] 7 Diazepam 13.9 285 257 1.90 10 [13.00–17.00] Midazolam 14.9 326 291 2.05 10 8 Prazepam 19.4 325 271 1.90 10 [17.00–21.00]

In spite of the extremely fast scan speed of the section of the Trap Control window) to an appro- Agilent LC/MSD Trap (up to 26,000 amu/s for priate percentage value of the precursor mass, or current models), configuring the MRM method to to “Default”, rather than setting a manual value for repetitively step through all of the precursor ions each analyte. The original version of this method and MS/MS scans for all 18 analytes plus internal used a manual cut-off value of 150 m/z for each standard would result in unacceptably long cycle MRM, which resulted in such a delay. Improved times and insufficient data points per second to sensitivity for these analytes is obtained by setting properly define and quantitate each analyte. the Cut-off to 45% rather than the default 27%. This Therefore the analysis is set up in time- essentially causes the trap to focus on the m/z programmed segments in which MRM occurs for regions where the major product ions of these ana- a few analytes at a time in a given portion of the lytes are found, not trapping lower mass ions less chromatogram, as shown in Table 3. useful for identification and quantitation.

A time delay may be observed during switchover The results of optimization of MS/MS acquisition between groups in which a large number of ana- can be seen by examining the chromatograms in lytes are being monitored if using manual Frag- Figure 1 which shows the overlaid principal prod- mentation Cut-off values. This delay can be uct ion chromatograms of all the analytes for the averted by setting the Cut-off selection in all analysis described here. groups (found under Fragmentation in the MS/MS

4 1.50 6

1. 7-aminonitrazepam 7. Flunitrazepam 13. N-desalkylfurazepam 1 2. 7-aminoclonazepam 8. Clobazam 14. Temazepam 3. 7-aminoflunitrazepam 9. Triazolam 15. Nordiazepam 1.25 4. Bromazepam 10. Alprazolam 16. Diazepam 5. Clonazepam 11. Lorazepam 17. Midazolam 6. Nitrazepam 12. Oxazepam

1.00 6 10

× 7

3 0.75 5 14 Intensity 8 12

0.50 10

2 9 13 16 0.25 4 17 15 11

0.00 4 6 8 10 12 14 Time [min]

Figure 1. Overlay of principal product ion chromatograms.

The first group, which extends from 1.0 to 4.0 min- Switching on the SmartFrag option may offer some utes, includes the MRM analysis of three com- advantages for qualitative analyses where spectral pounds, which practically co-elute. The second reproducibility and an abundance of product ions group only covers one compound, while the third are primary concerns. Switching off the SmartFrag group covers another three compounds. The fourth option to maximize the intensity of fewer product group covers six compounds, which reduces the ions will assist low level quantitative analyses. effective duty cycle for analyzing each compound. Whether or not the compounds are eluting, the Figures 2–8 show the structures and MS/MS spec- MRM is cycling through six different precursor tra for the analytes under the conditions of the ions. However, the chromatographic peak widths method. The spectra are grouped to illustrate some are large enough that sufficient data points are common losses and the interesting change in frag- produced for each analyte. mentation behavior that can occur with a relatively small change in structure. The product ion spectra are acquired in full scan mode which allows the MS/MS spectra to be added Figure 2 shows MS/MS spectra of the chlorine- to a user library as an automated aid to screening containing midazolam and triazolam which lose and compound identification. Such a library is in chlorine under these conditions. Triazolam with use in this laboratory and others in the toxicology the five-membered nitrogen-containing ring also field. For screening a larger number of drugs, the shows a major fragment ion corresponding to ring- AutoMSn mode of analysis can produce both MS opening and loss of diatomic nitrogen. and MS/MS (even MS/MS/MS, or MS3) spectra which can be searched against a library of spectra created using identical MSn parameters from authentic standards.

5 1.50 MS2(326.0), 14.3-15.6 min 291.1 Midazolam [M+H-Cl]+ N 1.25 H3C N

1.00 Cl N

5 F 10 × 0.75 Intensity

0.50

[M+H]+ 243.9 0.25 326.1 285.0 209.0 249.0 258.0 299.1 309.0 0.00 160 180 200 220 240 260 280 300 320 m/z

MS2(343.0), 7.4-8.3 min 308.1 [M+H-Cl]+ Triazolam 3 N H3C N N

N 5 Cl

10 2 × Cl

Intensity [M+H-N2]

1 315.0 [M+H]+

279.0 344.1 206.0 256.1 0 160 180 200 220 240 260 280 300 320 340 m/z

Figure 2. Structures and MS/MS spectra of midazolam and triazolam.

Figure 3 shows three benzodiazepines whose base different fragmentation: 7-aminoclonazepam loses peak in the MS/MS spectrum corresponds to loss HCl; 7-aminonitrazepam loses CO, apparently via of NO2. The figure also shows the spectra of the opening of the 7-membered ring; and 7-amino- metabolite of each parent drug in which the nitro flunitrazepam loses HF. Note the similarity in group has been reduced to an amino group. In structures for the analytes with HCl and HF losses. each case, the structural change gives rise to a

6 270.0 MS2(316.0), 5.8-6.3 min Clonazepam _ + [M+H NO2] 6 H O N

N O2N 5 Cl 10 4 ×

[M+H]+ Intensity

316.0 2

214.0 251.0 302.0 176.0 190.0 241.0 288.0 0 160 180 200 220 240 260 280 300 320 m/z 1.2 236.0 MS2(282.0), 6.0-6.5 min Nitrazepam _ + [M+H NO2] 1.0 H O N

0.8 N O2N 6 10 × 0.6 Intensity

0.4 [M+H]+

0.2 282.0 254.0 180.0 208.0 176.0 268.0 0.0 160 180 200 220 240 260 280 m/z

6 268.1 MS2(314.0), 6.1-6.5 min Flunitrazepam _ + [M+H NO2] 5 CH 3 O N

4 N 5 O2N 10

× F 3

Intensity 286.1

2 [M+H]+

240.0 314.1 1 193.0 211.0 257.0 300.1 228.0 0 160 180 200 220 240 260 280 300 320 m/z

Figure 3. Structures and MS/MS spectra for clonazepam, nitrazepam, flunitrazepam, 7-aminoclonazepam, 7-aminonitrazepam, and 7-aminoflunitrazepam.

7 1.2

250.0 MS2(286.0), 2.5-3.9 min 7-aminoclonazepam [M+H_HCl]+ 1.0 H O N 0.8 N 6 H2N 10

× Cl 0.6 Intensity

0.4 [M+H]+ 286.1 222.0

0.2

0.0 160 180 200 220 240 260 280 300 320 m/z

224.0 MS2(252.0), 2.1-3.2 min

_ + 3 7-aminonitrazepam [M+H CO] H O N 5

10 2

× N H2N 253.1 Intensity

1 207.0 [M+H]+

180.0 234.0 195.0 173.9 189.9 217.0 0 160 180 200 220 240 260 m/z MS2(284.0), 2.8-3.7 min 264.1 6 _ + 7-aminoflunitrazepam [M+H HF] 256.1 CH 3 O N

5 4 10 H N N 227.0 × 2 236.0 F 163.0 Intensity [M+H]+

2 284.1 207.0

215.0 269.0 180.9 219.0 0 160 180 200 220 240 260 280 m/z

Figure 3 (continued). Structures and MS/MS spectra for clonazepam, nitrazepam, flunitrazepam, 7-aminoclonazepam, 7-aminonitrazepam, and 7-aminoflunitrazepam.

8 Flurazepam and prazepam lose alkyl groups as substituent develops a different fragmentation shown in Figure 4, and the desalkylflurazepam behavior with the major ion corresponding to loss metabolite which has lost the entire alkylamino of CO.

1.25 315.1 AII, 7.3-8.5 min

_ + Flurazepam [M+H NH(C2H5)2]

1.00 CH2CH2N(C2H5)2 O N 6 0.75 Cl N F Intensity x10

0.50

[M+H]+

0.25 388.2 288.0

0.00 150 175 200 225 250 275 300 325 350 375 400 m/z

261.0 226.0 MS2(289.0), 9.0-9.6 min N-desalkylflurazepam [M+H_CO]+ 2.0 H O N

1.5 Cl N 5 F

Intensity x10 1.0 290.0 [M+H]+ 164.9 0.5 214.0

192.9 205.0 236.0 254.0 0.0 160 180 200 220 240 260 280 300 m/z

271.0 6 Prazepam MS2(325.2), 7.4-7.9 min, + CH [M+H-C4H6] 5 2 O N

5 4 N Cl 3 Intensity x10 [M+H]+

2 325.0

1 254.8

0 160 180 200 220 240 260 280 300 320 340 m/z

Figure 4. Structures and MS/MS spectra for flurazepam, N-desalkylflurazepam, and prazepam.

9 Figure 5 shows the loss of N2 from alprazolam under these conditions. Its structure is very simi- lar to that of triazolam (Figure 2) which also loses

N2, presumably also from the five-membered ring.

MS2(309.0), 7.8-8.6 min 4 Alprazolam 281.1 [M+H_N ]+ N 2 H2C N N 3

Cl N

5 274.1

2 Intensity x10

[M+H]+ 1 241.0 309.1 206.0 251.0 165.0 216.0 226.0 0 160 180 200 220 240 260 280 300 320 m/z

Figure 5. Structure and MS/MS spectrum of alprazolam.

Figure 6 shows several more benzodiazepines which lose the elements of CO, like N-desalkylflurazepam in Figure 4. It is interesting that all three lose CO from the 7-ring even though they all have a halogen in the 7-position of the fused benzene ring. Notice the HX loss from 7-aminoclonazepam and 7-aminoflunitrazepam (Figure 3) where a halogen is on the 2'-position of the non-fused benzene ring.

10 Bromazepam 288.0 MS2(316.0), 5.1-5.5 min 1.50 H O N [M+H_CO]+ 1.25 209.0 Br N

5 1.00 N

0.75 182.0 [M+H]+ Intensity x10

260.9 0.50 316.0

0.25 237.0 298.0 0.00 160 180 200 220 240 260 280 300 320 m/z

2.0 257.0 MS2(285.0), 13.3-14.4 min Diazepam [M+H_CO]+ CH3 O N 1.5 Cl N 5 222.0 1.0 Intensity x10 228.0 [M+H]+ 193.0 154.0 181.9 0.5 285.1

166.9 203.9 0.0 160 180 200 220 240 260 280 m/z 1.50 Nordiazepam 208.0 MS2(271.0), 11.7-12.6 min

H _ + 1.25 O [M+H CO] N 243.0

1.00 Cl N 5

165.0 0.75 226.0 Intensity x10

0.50

+ 0.25 192.9 [M+H] 272.0 213.9 234.9 180.0 252.9 0.00 160 180 200 220 240 260 m/z

Figure 6. Structures and MS/MS spectra for bromazepam, diazepam, and nordiazepam.

11 Clobazam shows an extremely simple MS/MS spectrum and a unique loss of CH2CO in Figure 7.

MS2(301.0), 7.1-8.1 min Clobazam 259.0

8 CH3 O _ + N [M+H CH2CO]

Cl N 6 O 5

4 [M+H]+ Intensity x10

301.1 2

0 160 180 200 220 240 260 280 300 320 m/z

Figure 7. Structure and MS/MS spectrum for clobazam.

Figure 8 shows the MS/MS spectra of the remain- ing benzodiazepines which are obtained using an important feature of the Agilent LC/MSD Trap. On initial examination of MS/MS spectra during method optimization, lorazepam, oxazepam, and temazepam were found to have the major product ion to be the result of loss of the elements of water. As this is not the most specific loss one might prefer for identification purposes, a more information-rich MS/MS spectrum was obtained for each using the following technique. By increasing the fragmentation window from 10 amu to 40 amu (±20 amu centered on the precursor ion mass), fragmentation energy is applied to both the [M+H]+ + and the [M+H–H2O] ions, and the resulting MS/MS spectra are much more specific for identification.

12 275.0 MS2(321.0), 7.8-8.6 min Lorazepam 1.5 H O _ _ + N [M+H H2O CO] OH Cl N 5 1.0 Cl Intensity x10

+ 0.5 [M+H]

321.1 303.0

0.0 160 180 200 220 240 260 280 300 320 m/z 5 241.0 MS2(287.0), 7.9-8.9 min Oxazepam _ _ + H [M+H H2O CO] 4 O N OH Cl N 5 3

Intensity x10 2

[M+H]+

1 287.0

269.0 0 160 180 200 220 240 260 280 300 m/z 5 255.0 MS2(301.0), 9.2-10.1 min Temazepam

CH3 4 O _ _ + N [M+H H2O CO] OH Cl N 5 3

Intensity x10 2 [M+H]+

1 301.0

216.0 165.9 273.0 0 160 180 200 220 240 260 280 300 m/z

Figure 8. Structures and MS/MS spectra for lorazepam, oxazepam, and temazepam.

13 For example, with temazepam, fragmentation can Table 4. Case Examples of Drugs and their Concentrations result in the water-loss ion at m/z 283.0 and the in Blood water-loss plus CO loss ion at m/z 255.0. If the Case Benzodiazepine/ Concentration fragmentation energy is applied not only to the metabolite mg/L pseudomolecular ion at m/z 301.0, but also to the 1 Nitrazepam 0.01 water-loss ion by using a fragmentation window of 7-aminonitrazepam 0.08 40 amu, the intensity of the m/z 255.0 is improved, Diazepam 0.33 resulting in better detection and identification. Nordiazepam 0.32

Application to Forensic Cases 2 Diazepam 0.06 Clonazepam 0.009 Blood extracts from a wide variety of case types 7-aminoclonazepam 0.02 including multiple drug overdose, sexual assault 3 Bromazepam 0.40 victims and motor vehicle accident victims have been analyzed by this LC/MS/MS procedure. A 4 Alprazolam 0.006 number of benzodiazepines have been identified Diazepam 0.05 Nordiazepam 0.01 using this screening method, and are subsequently quantified by GC/ECD or HPLC-UV. A selection of 5 Diazepam 0.58 the cases and their blood drug concentrations are Nordiazepam 0.66 shown in Table 4. Oxazepam 0.04 Temazepam 0.12 The ion chromatograms from the blood sample in 6 Clobazam 0.10 Case 1, which provide the detection of nitrazepam, 7-aminonitrazepam, diazepam, nordiazepam and prazepam, are shown in Figure 9.

BENZ0006.D: EIC 224 ±MS2(252.1), Smoothed (3.2,1, GA) (mg/L 4 4 Compounds or µg/mL) 3 2.6 min 7-aminonitrazepam 7-NH2-nitrazepam 0.08 2 1 Intensity x10 0 6

4 5 BENZ0006.D: EIC 236 ±MS2(282.1), Smoothed (3.1,1, GA) 4 Nitrazepam 0.01 3 6.2 min nitrazepam 2

Intensity x10 1 0 2.0

5 BENZ0006.D: EIC 243 ±MS2(271.1), Smoothed (2.7,1, GA) 1.5 Nordiazepam 0.32 1.0 12.2 min nordiazepam 0.5 Intensity x10 0.0

4 BENZ0006.D: EIC 257 ±MS2(285.1), Smoothed (4.1,1, GA) 5 3 14.0 min diazepam Diazepam 0.33 2 1

Intensity x10 0 1.5 BENZ0006.D: EIC 271 ±MS2(325.2), Smoothed (3.1,1, GA) 7 1.0 19.4 min prazepam Prazepam (internal standard) 0.5 Intensity x10 0.0 20 4 6 8 10 12 14 16 18 20 Time [min]

Figure 9. Ion chromatograms from Case 1 example.

14 Polypharmacy in such cases is not uncommon, and the method can easily detect, confirm and quantify multiple benzodiazepines and metabolites in a single analysis, as illustrated by this case.

Typical ion chromatograms from a blank blood sample are shown in Figure 10. Note that in Case 4 (see Table 4) it was possible to detect 0.006 mg/L (6 ng/mL) of alprazolam while still obtaining a clear identification with a full-scan MS/MS spectrum. Case 4 contains the lowest level of benzodiazepine detected in these cases, that of alprazolam at a level of 6 µg/L (6 ng/mL).

4000 DS030822\BENZ0007.D: EIC 224 ±MS2(252.1) 3000 2000

Intensity 1000 0

1500 DS030822\BENZ0007.D: EIC 236 ±MS2(282.1) 1000 500 Intensity 0

DS030822\BENZ0007.D: EIC 243 ±MS2(271.1) 3000

2000

1000 Intensity 0

DS030822\BENZ0007.D: EIC 257 ±MS2(285.1) 3000 2000 1000 Intensity 0

DS030822\BENZ0007.D: EIC 271 ±MS2(325.2) 1.5 7 1.0 19.4 min prazepam 0.5

Intensity x10 0.0 02 4 6 8 10 12 14 16 18 20 Time [min]

Figure 10. Ion chromatograms from blank blood with internal standard.

15 www.agilent.com/chem Conclusions

The LC/MS/MS method described here provides a single procedure for the identification of a wide range of benzodiazepines available in Australia and their metabolites, with a simple adaptation of an existing GC/MS sample preparation procedure, and without the need for derivatization. The MS/MS spectra provide a high-confidence identification of the drugs. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported dosage oncentrations using only 500 µL of blood. The data in Table 4 and Figure 9 illustrate that the procedure is able to identify a range of dose concentrations of benzodiazepines in casework samples. Low concentrations of various benzodiazepines have been rapidly and successfully identified in forensic cases.

References

1. Y. Gaillard, J. Gay-Montchamp and M. Ollagnier,(1993) J. Chromatogr. 622, 197. 2. D. Black, G. Clark, V. Haver, J. Garbin and A. Saxon, (1994) J. Anal. Toxicol. 18, 185. 3. M. Elsohly, S. Feng, S. Salomone and R. Brenneisen, (1999) J. Anal. Toxicol. 23, 486. 4. I. McIntyre, M. Syrjanen, K. Crump, S. Horomidis, A. Peace and O. Drummer, (1993) J. Anal. Toxicol. 17, 202. Acknowledgements Special thanks are due to the Australian authors for sharing their method, and for 5. C. Kratzsch, O. Tenberken, F. T. Peters, their patience during the preparation of this note. Thanks are due also to Agilent A. A. Weber, T. Kraemer and H. H. Maurer, colleagues Michael Zumwalt for encouragement and a first draft, and to Jeff Keever (2004) J. Mass Spectrom. 39, 856–872. for review and helpful comments.

6. H.M. Rivera, G. S. Walker, D. N. Sims, and P.C. Stockham, (2003) European Journal of For More Information Mass Spectrometry, 9, 599–607. For more information on our products and services, visit our Web site at www.agilent.com/chem. For more details concerning this note, please con- tact John Hughes at Agilent Technologies, Inc.

For Forensic Use only.

Information is subject to change without notice.

Printed in the USA March 7, 2006 5989-4737EN