Authors

Fredrick D. Foster, John R. Stuff, Edward A. Pfannkoch;
GERSTEL, Inc.,
701 Digital Dr. Suite J,
Linthicum, MD 21090,
USA.

Sparkle T. Ellison, William E. Brewer, Stephen L. Morgan;
Department of Chemistry and Biochemistry,
University of South Carolina,
631 Sumter Street,
Columbia, SC 29208,
USA.

Tom Gluodenis;
Agilent Technologies,
2850 Centerville Rd.,
Wilmington, DE 19808,
USA.

For more information please contact us by e-mail: gerstel@gerstel.com

Urine Sample Pretreatment Whole Blood Sample Pretreament
  • Pipette 260μL of hydrolyzed urine sample into a clean 12 x 75mm culture tube.
  • Pipette 250μL of 1.0M HCl into the tube and vortex mix for a few seconds.
  • Pipette 250μL of acetonitrile into the tube and vortex mix for a few seconds.
  • Filter the sample using an Agilent 2 in 1 syringe filter and collect the filtrate into a clean 12 x 75mm culture tube.
  • Place the filtered urine sample onto the GERSTEL MPS 2XL multi-purpose sampler with DPX Option.
  • Pipette 200μL of whole blood sample into a clean 12 x 75mm culture tube.
  • Pipette 800μL of acetonitrile into the tube and vortex mix for a few seconds.
  • After centrifugation for 10 minutes to pellet the precipitated proteins, transfer the supernatant into a clean 12 x 75mm culture tube.
  • Place the sample onto the GERSTEL MPS 2XL multi-purpose sampler with DPX Option.
 Analyte Precursor Ion [m/z] Product Ions [m/z] Fragmentor Voltage [V] CE (V)
 bromazepam 316 182.1 (209) 140 (140) 30 (25)
 nitrazepam 282 236 (180) 160 (160) 25 (35)
 d4-clonazepam 320 274,1 120 30
 flunitrazepam 314 239 (268) 160 (160) 35 (30)
 clonazepam 316 270 (214) 120 (120) 25 (30)
 d5-a-OH-alprazolam 330 302 120 30
 d5-estazolam 300 272 140 25
 a-OH-alprazolam 325 216 (297) 120 (120) 35 (30)
 estazolam 295 205 (267) 160 (160) 40 (25)
 clobazam 301 224 (259) 140 (140) 30 (15)
 triazolam 343 239 (308) 180 (180) 40 (25)
 alprazolam 309 274 (281) 160 (160) 30 (25)
 d5-oxazepam 292 246 120 20
 oxazepam 287 241 (269) 120 (120) 20 (15)
 lorazepam 321 229 (275) 140 (140) 30 (20)
 temazepam 301 255 (177) 120 (120) 35 (40)
 d5-nordiazepam 276 213 160 30
 nordiazepam 271 140 (165) 160 (160) 30 (30)
 midazolam 326 249 (291) 180 (180) 40 (25)
 diazepam 285 222 (257) 160 (160) 25 (25)
 flurazepam 388,1 288 (315) 140 (140) 25 (20)
Mass Spectrometer Acquisition Parameters
   
 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
 a-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).
  Compound Recovery [%]
  Alprazolam 65
  a-OH-alprazolam 100
  Clonazepam 91
  Diazepam 80
  Flunitrazepam 86
  Lorazepam 49
  Nitrazepam 93
  Nordiazepam 91
  Oxazepam 91
  Temazepam 84
  Estazolam 90
  Clobazam 6
  Triazolam 54
  Flurazepam 82
  Midazolam 57
  Bromazepam 43
Recovery for 16 benzodiazepines using our DPX-LC/MC/ MS-Method. The low recovery of clobazam is based on its low extraction efficiency. Unlike the other benzodiazepines it does not contain a tertiary amine in its chemical structure and therefore binds less tightly to the DPX-CX cation exchange material. This result supports the specifity of the DPX-CX sorbent for tertiary amines.

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.

 

 
 Compound   QC1 QC2 QC3
    7.5ng/mL 30ng/mL 75ng/mL
 a-OH-alprazolam mean 7,50 29,8 78,7
  %RSD 4,30 1,40 2,31
  %Accuracy 101 99,3 105
  N 6 5 6
 estazolam mean 7,31 30,5 80,6
  %RSD 1,52 1,01 1,55
  %Accuracy 99,3 102 108
  N 6 5 6
 lorazepam mean 6,04 29,5 91,4
  %RSD 8,97 8,48 11,5
  %Accuracy 89,3 98,3 122
  N 6 5 6
 oxazepam mean 7,21 30,3 79,6
  %RSD 1,74 1,68 1,22
  %Accuracy 98,6 101 106
  N 6 5 6
 temazepam mean 6,97 30,3 78,5
  %RSD 1,96 2,29 1,72
  %Accuracy 97,2 101 105
  N 6 5 6
 alprazolam mean 6,65 29,4 86,8
  %RSD 5,03 4,25 3,24
  %Accuracy 93,7 97,9 116
  N 6 5 6
 clobazam mean 7,54 31,6 66,4
  %RSD 13,7 5,67 15,3
  %Accuracy 101 105 88,5
  N 6 5 6
 clonazepam mean 7,30 29,3 79,0
  %RSD 1,91 2,05 1,75
  %Accuracy 97,7 97,5 105
  N 6 5 6
 
 Compound   QC1 QC2 QC3
    7.5ng/mL 30ng/mL 75ng/mL
 diazepam mean 6,96 30,1 83,7
  %RSD 3,67 3,95 2,19
  %Accuracy 97,7 100 112
  N 6 5 6
 flunitrazepam mean 7,33 29,9 80,4
  %RSD 1,67 2,85 1,36
  %Accuracy 98,5 100 107
  N 6 5 6
 flurazepam mean 6,15 27,8 89,0
  %RSD 9,96 10,3 5,25
  %Accuracy 86,6 92,5 119
  N 6 5 6
 midazolam mean 6,02 27,7 87,9
  %RSD 9,12 9,37 7,40
  %Accuracy 84,9 92,4 117
  N 6 5 6
 nitrazepam mean 7,15 29,5 80,2
  %RSD 2,17 4,59 1,44
  %Accuracy 97,6 98,4 107
  N 6 5 6
 nordiazepam mean 7,38 29,3 77,8
  %RSD 2,63 2,10 1,77
  %Accuracy 99,5 97,8 104
  N 6 5 6
 triazolam mean 6,13 27,0 81,7
  %RSD 6,46 3,69 9,79
  %Accuracy 86,7 89,9 109
  N 6 5 6
 bromazepam mean 8,20 31,0 102,0
  %RSD 14,8 15,5 26,4
  %Accuracy 104 103 136
  N 6 5 6
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%).