Compound  R² LOQ [ng/mL]  temazepamC 0.9917 0,5  alprazolamC 0.9901 0,5  oxazepamC 0.9950 0,5  lorazepamC 0.9908 0,5  estazolamD 0.9955 0,5  α-OH-alprazolamA 0.9958 0,5  flurazepamB 0.9937 0,5  midazolamB 0.9919 0,5  flunitrazepamE 0.9959 0,5  diazepamB 0.9969 0,5  clonazepamE 0.9974 0,5  clobazamC 0.9947 0,5  bromazepamE 0.9973 0,5  triazolamC 0.9783 0,5  nordiazepamB 0.9941 0,5  nitrazepamC 0.9910 0,5

Representative Calibration Curve of Triazolam and Clonazepam.

Calibration Curve Results Calculated using d5-a-Hydroxyalprazolam (A), d5-Nordiazepam (B), d5-Oxazepam (C), d5-Estazolam (D) and d4-Clonazepam (E).

Automated Sample Preparation

Doing Drugs? II

Comprehensive Analysis of Drugs of Abuse in Urine with Automated Disposable Pipette Extraction (DPX) and HPLC/MS/MS with dynamic MRM.

In order to analyze biological specimens for drugs such as benzodiazepines, it is necessary to perform sample preparation to eliminate matrix interference and ion suppression. Solid-phase extraction is generally the preferred sample preparation technique, in this study Disposable Pipette Extraction (DPX) was utilized. DPX is a fast dispersive solid-phase extraction technique that uses loosely contained sorbent in a disposable pipette tip. The sample is aspirated into the tip where it is actively mixed with the sorbent and forms a suspension. The main advantages of the DPX technology are that the extraction is rapid, minimal solvent waste is generated, and the entire process can be fully automated including introduction of the extract to the chromatographic system. The GERSTEL MPS autosampler performs DPX extractions in approximately 5 minutes using reversed phase (DPX-RP), cation exchange (DPX-CX) or immunoaffinity sorbent material. For determination of target drugs, GC/MS or HPLC/MS/MS are generally the preferred techniques. The advantage of LC/MS/MS is that chemical derivatization of the analytes is not required, making sample preparation simpler and less time consuming. In addition, highly efficient ionization, in combination with tandem mass spectrometry results in high sensitivity and selectivity. This study focused on performing automated extraction of reduced sample volumes coupled with LC/MS/MS to provide high throughput analysis “one sample at a time”. The sample preparation time was decreased sufficiently to allow the extraction of a sample during the chromatographic analysis of the previous sample in the sequence.

Experimental

Instrumentation: All Analyses were performed using an Agilent 1200 HPLC with an Agilent 6410 Triple Quadrupole Mass Spectrometer with ESI source. Sample Preparation and Sample Introduction was performed using a GERSTEL MultiPurpose Sampler (MPS XL).

Materials

Benzodiazepine Multi-Component Mixture 8, containing Clonazepam, Temazepam, Nitrazepam, Alprazolam, Diazepam, Flunitrazepam, Lorazepam, and Oxazepam at 250 μg/mL each in acetonitrile, Nordiazepam, Clobazam, Bromazepam, Estazolam, Flurazepam, Midazolam, and Triazolam, at 1.0 mg/mL each in methanol, and α-hydroxyalprazolam, at 100 μg/mL in methanol, were purchased from Cerilliant. Intermediate stock solutions of the sixteen benzodiazepines were prepared in water from appropriate dilutions of these stocks. Deuterated analogues d5-nordiazepam, d5-α-hydroxyalprazolam, d5-oxazepam, d4-clonazepam, and d5-estazolam, at 100 μg/mL each in methanol, were purchased from Cerilliant. These stocks were combined and diluted with water to be used as internal standards during analysis.

Extraction

A GERSTEL MultiPurpose Sampler (MPS) was set up with 1 mL DPX-CX tips (DPX Labs, LLC, Columbia, SC) for extraction of hydrolyzed urine samples. The following automation method was used: 250 μL of 30% acetonitrile/water was slowly added through the top of the DPX tip at a rate of 50 μL/s to wet the sorbent. The sample was then aspirated into the DPX tip and mixed with the sorbent by drawing in an additional 1.3 mL of air. After a 20 s equilibration time to allow analyte binding, the resulting solution was dispensed to waste. To wash off excess matrix, 500 μL of a 10% acetonitrile/water wash solution was added to the sorbent material through the top of the DPX tip and dispensed to waste followed by an additional wash using 500 μL of 100% acetonitrile. For elution of the analytes, 700 μL of 78/20/2 (v/v) of methylene chloride/isopropanol/ammonium hydroxide was added to the sorbent material through the top of the DPX tip and the eluate dispensed directly into a clean 2 mL autosampler vials. All eluents were dried and reconstituted with 50 μL of water before injection.

Results and Discussion

The DPX-CX extractions were readily performed using the GERSTEL MultiPurpose Sampler. The entire extraction process took approximately 6.5 minutes per sample. Because a basic eluent is used with the cation exchange sorbent, the eluents had to be solvent exchanged into the HPLC mobile phase. In light of the world-wide acetonitrile shortage, it was found that the use of 10%  isopropyl alcohol in methanol as the organic modifier of the mobile phase provided an alternative to using acetonitrile. All HPLC/MS spectra were collected using dynamic multiple reaction monitoring (MRM) providing good sensitivity for the analysis of these drugs at low concentrations. A rapid resolution HPLC column was chosen in order to perform the chromatographic data in approximately 10 minutes.

Extracted Ion Chromatogram of DPX extracts of urine samples spiked at 7.5 ng/mL

Conclusion

Automated DPX extraction of benzodiazepines from urine can be performed successfully using the GERSTEL MPS. In the work presented here, the total extraction time was 6.5 minutes, The total sample preparation time was less than the chromatographic run time, which means that the next sample can be prepared while separation of the current sample is in progress. Whenever the LC/MS/MS system has finished a run, the next sample is ready to be introduced ensuring the highest possible throughput. Additionally, “just in time” sample preparation helps to ensure that the prepared sample is not kept in the autosampler for a long time prior to being analyzed, reducing the risk of analyte degradation and helping to maintain sample integrity.
Using this DPX-LC/MS/MS method for determination of benzodiazepines in urine high recoveries for all benzodiazepines were achieved. The calibration curves showed good linearity (R2 ≥ 0.98) with limits of quantitation of 0.5 ng/mL. The method proved to be accurate and precise. Accuracy data averaged 102% (range: 84.9% - 136%) and precision data averaged 5.48%RSD (range: 1.01% -26.4%) for all benzodiazepines analyzed.

QC samples accuracy and precision table

Matrix effects on the ionization of benzodiazepines in hydrolyzed urine and whole blood compared to the neat standard solutions in pure water (100%).