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Identifying Benzodiazepines

Michael Zumwalt and John M Hughes narrate the use of LC/MS in tracing Benzodiazepines.

The analysis of benzodiazepines is of great interest to forensic and clinical toxicologists. Using the Agilent LC/MSD VL Quadrupole mass spectrometer instrument in full scan mode, both spectral identification and quantitation can be carried out simultaneously.

Benzodiazepines are an important class of drugs with a broad range of therapeutic effects, including sedative-hypnotic, anxiolytic, muscle-relaxant, and anticonvulsant. Because of their wide usage, benzodiazepines have the potential for interaction with other central nervous system depressants which can result in life-threatening or impaired driving situations. Benzodiazepines are now among the most commonly-prescribed drugs, which increases their potential for addiction and abuse, and often they are found in combination with other drugs in drug-related fatalities or drug facilitated sexual assault cases. The analysis of benzodiazepines is therefore of great interest to forensic and clinical toxicologists.

Benzodiazepines have been analysed using HPLC with UV detection, gas chromatography with nitrogen phosphorus and electron capture detectors, and gas chromatography/mass spectrometry (GC/MS). Many benzodiazepines are polar and non-volatile, making them difficult, if not impossible, to analyse with GC or GC/MS. Some of the compounds cannot be derivatised for improved chromatographic behaviour. Furthermore, some of the newer benzodiazepines, like flunitrazepam, have lowered therapeutic ranges and faster clearance, and therefore require quantitation at lower levels. Liquid chromatography/quadrupole mass spectrometry is ideally suited for these compounds because the technique does not require derivatisation, thereby saving time, expense, and experimental difficulty. The full-scan sensitivity of the Agilent liquid chromatograph/mass selective detector (LC/MSD) allows for quantitation, identification and confirmation in a single analysis.

Experimentally...

The LC/MS system consists of 1100 series vacuum degasser, binary pump, autosampler, thermostatted column compartment, diode array detector (DAD) with micro-flow cell, and LC/MSD quadrupole VL model. The DAD was used primarily for method development. However, the UV detector in series with the MS provides UV spectra which can also be used for identification when levels are sufficiently high. Complete system control and data analysis was provided by the Agilent LC/MS ChemStation.

Sample preparation

Samples were prepared using liquid-liquid extraction, which is commonly used for these compounds for analysis by GC/MS. The only difference from a GC/MS method is omitting the derivitisation step and reconstitution of the final sample in the LC mobile phase, rather than in a volatile solvent for GC injection. A 1 ml volume of blood, serum, or plasma, to which 100 µL of internal standard solution (10 ng/µL) has been added, is added to 1 ml of saturated sodium borate solution, and the mixture is vortex-mixed. Ethyl acetate (4 ml) is added and mixing is carried out on a rotary shaker for five minutes, followed by centrifugation at 3400 rpm for five minutes. The upper layer is transferred to a clean tube and evaporated to dryness. The residue is reconstituted in 50 µl of the initial mobile phase and transferred to an autosampler vial.

Results and discussion

The sample preparation used for many GC or GC/MS methods can often be used for LC/MS just by omitting any derivatisation step and transferring the final sample to LC mobile phase instead of using a volatile solvent. For this experiment, Flumazenil, a benzodiazepine antagonist not found in samples in this jurisdiction, was used as the internal standard due to the cost and availability of deuterated analogues of some analytes.

The standard VL model of the LC/MSD is quite capable of carrying out the analysis of benzodiazepines in blood. The SL model affords approximately 10x greater sensitivity if needed for other analyses, as well as multisignal capability such as alternating positive/negative mode, SIM/scan mode, and low/high fragmentation modes. The analysis was carried out in full scan acquisition mode in order to quantitate the target analytes using extracted ion chromatograms (EICs), and to alternatively confirm their identity using other ions in the spectra as well.

A moderate amount of collision-induced dissociation (CID) was used in this method by setting the fragment or voltage in the method to a value 50V higher than the default value of 70V which minimises CID. This results in spectra which contain more ions than just the pseudo-molecular ion. These ions can then be used as confirming ions for EICs, as is the common practice for EI GC/MS, and the spectra can be placed in a user created library for identification of drugs using library search of API spectra.

The fragmentor voltage chosen is just high enough to produce fragment ions by CID for confirmation, while attempting to preserve significant signal for the intact molecule. For example, the m/z 268.1 in the spectrum of flunitrazepam arises from fragmentation of the (M+H)+ ion at m/z 314.1. The spectral behaviour is, of course, compound dependent. For instance, in the case of temazepam, there is more signal for the sodium adduct at m/z 323.1 so that ion is used for quantitation, while the fragment at m/z 255.0 is used for confirmation. The protonated molecular ion m/z 301.0 can also be used as a confirmation ion, as long as the protonated/sodiated ion ratios are constant for the analysis. Compounds with oxygen-containing functional groups can show sodium adducts as well as proton adducts; this complication can be avoided with the use of Atmospheric Pressure Chemical Ionisation (APCI) in place of electrospray ionisation (ESI).

The limit of detection (LOD) at a S/N of 3:1 is approximately 10 ng/ml using this method for most of the target compounds and this model of LC/MSD. The method as practised at the Montana State Toxicology laboratory uses a 20 ng/ml (0.02 µg/ml) limit of quantitation (LOQ), a calibration range extending to 1,000 ng/ml, and one or
more qualifier ions for each analyte.

With the specified sample preparation and instrument conditions and a calibration range up to 1000 ng/mL, a quadratic treatment gives a better curve fit than a linear treatment for most of these analytes (r2 >0.99). Results of an actual case sample, in which alprazolam was found at a moderate level of 128 ng/ml.

Conclusion

This experiment demonstrated the usefulness of LC/MS for the analysis of benzodiazepines in blood. These compounds tend to be difficult to analyse by GC-based techniques, but ionise well in API-electrospray, resulting in excellent sensitivity, even in full scan mode using the lowest-cost model of LC/MSD (VL). Blood is a difficult matrix to analyse, but the results here show excellent quantitation and simultaneous identification using only 1 ml of sample and a simple liquid- liquid extraction procedure used for GC/MS, without the derivatisation. The CID spectra show strong basepeak signals used for quantitation over three orders of magnitude, and CID fragment ions for ion ratio confirmation and/or library search. The method could be used in SIM and with the more sensitive LC/MSD SL model for any of the newer benzodiazepines which are found at lower levels in blood.

A rapid, validated method has also been published for a large number of benzodiazepines and related substances using the Agilent LC/MSD quadrupole system. The method uses APCI rather than ESI, a liquid/liquid extraction procedure similar to this one, CID with greater fragmentation, and several deuterated internal standards. The publication describes the use of CID spectra and library search for identification, and includes spectra of all the analytes under both low and high fragmentation conditions.

(The authors are Senior Application Scientist and Senior Applications Consultant, Mass Spectrometry of Agilent)