Authors

Oscar G. Cabrices, Fred D. Foster, John R. Stuff, Edward A. Pfannkoch,
GERSTEL, Inc., 701 Digital Dr. Suite J, Linthicum, MD 21090, USA
William E. Brewer
Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA

Figure 1: MPS XL MultiPurpose Sampler (dual head version) with GERSTEL DPX option, mounted on top of an Agilent 6460 Triple Quad LC/MS system, for high throughput pain management drug screening.
Figure 2: Graphical representation of the automated DPX urine cleanup process. The new extraction procedure described here, combines the advantages of dispersive SPE and liquid-liquid extraction in a simple, quick and efficient manner. Therefore, only one extraction step is needed to eliminate the whole range of potentially interfering matrix compounds. As can be seen, an ACN layer and an aqueous layer are formed thanks to the salts contained in the DPX tip.
Figure 3: Overlaid chromatograms for all 116 dynamic MRM transitions from an extracted urine sample at the MRL.
Analysis conditions LC
Pump: gradient (600 bar),
flowrate = 0.5 mL/min 
Mobile phase: A - 5 mM ammonium
formate, with 0.05 % formic acid
B - 0.05 % formic acid in methanol
Gradient: Initial 5 % B
0.5 min 5 % B
1.5 min 30 % B
3.5 min 70 % B
4.5 min 95 % B
6.49 min 95 % B
6.5 min 5 % B
Run time: 6.5 minutes
Injection volume: 2 µL (loop over-fill technique)
Column temperature: 55 °C
Analysis conditions MS
Operation: electrospray positive ion mode
Gas temperature: 350 °C
Gas flow (N2): 12 L/min
Nebulizer pressure: 35 psi
Capillary voltage: 4400 V 
Figure 4: Representative calibration curves: morphine, flurazepam, cocaine and ketamine.

Drug screening

No pain!

This study focuses on high throughput automated extraction of small volumes of urine samples (< 500 μL) used in the determination of pain management drugs by LC-MS/MS. Disposable pipette extraction (DPX) was used in a novel manner (see figure 2) to extract pain management drugs for comprehensive screening. Extracts were automatically diluted and injected into the LC-MS/MS system. Sample preparation was performed “just-in-time”, the cycle time averaged 7 min per sample. Validation results show that the automated DPX-LC-MS/MS screening method provides adequate sensitivity for more than 65 analytes and internal standards. Lower limits of quantitation (LLOQ) ranged between 0.5 – 50 ng/mL and % RSDs were below 10 % in most cases.

Toxicology laboratories are trying to find ways to minimize sample preparation and enhance productivity. The adaptation of LC-MS/MS instrumentation has become popular due to the technique’s high sensitivity and selectivity, low detection limits (e.g. 1 ng/mL), smaller sample volume requirements, and also due to the fact that LC-MS/MS doesn’t require chemical derivatization of analytes. However, LC-MS/MS can require the use of sample clean-up, extraction and concentration steps. These steps have traditionally been performed manually using liquid-liquid or solid-phase extraction (SPE).
A different approach is to use SPE to extract the sample matrix. In this case, matrix interferences are bound to the sorbent in order to be removed from the analyte solution. The major advantage of this approach is that no separate wash or elution steps are required, enabling rapid sample preparation while still allowing comprehensive screening of the cleaned sample. Disposable Pipette Extraction (DPX) was developed as an alternative to traditional SPE, combining efficient and rapid extraction with significantly reduced solvent consumption. DPX is a novel dispersive solid-phase extraction technique that uses sorbent loosely contained in a pipette tip enabling highly efficient mixing with the sample solution. The main advantages of the DPX technology are: rapid extraction, high recoveries, negligible solvent waste generation, and full automation of the extraction combined with sample introduction to the chromatographic system. We have developed a fast automated DPX urine cleanup method using a GERSTEL MultiPurpose Sampler (MPS XL) for comprehensive screening of 49 pain management drugs with LC-MS/MS. The reversed phase sorbent with added salts (DPX-RPS) used in the method allows the removal of salts and proteins present in urine, resulting in reduced matrix effects. The novel DPX procedure described here combines the advantages of dispersive SPE and liquid extraction in a simple, quick and efficient way.
The sorbent is chosen to extract the matrix without binding or absorbing the analytes of interest providing high recoveries. Since the extraction time (3 min) is less than the analytical LC-MS/MS run time (4 min), the extraction of one sample can be performed during the chromatographic analysis of the previous sample, achieving high throughput while processing each sample “just in time” ensuring that all samples are treated identically.

EXPERIMENTAL

Materials.Stock solutions for the compounds listed in Table 1 were purchased from Cerilliant. An intermediate analyte stock solution was prepared by combining the analyte stock solutions with acetonitrile, at appropriate concentrations, to evaluate the different drug classes.
Deuterated analogues, d3-morphine, d4-buprenorphine, d3-norbuprenorphine, d9-methadone, d3-tramadol, d5-fentanyl, d5-alpha-hydroxy alprazolam, d4-clonazepam, d5-oxazepam, d5-estazolam, d3-cocaine, d5-nordiazepam, d5-propoxyphene, d7-carisoprodol, d5-amphetamine, d4-ketamine, d4-7-aminoclonazepam, and d5-PCP were purchased from Cerilliant. High concentration calibration standard and intermediate QC urine samples were prepared by making appropriate dilutions of the combined intermediate analyte stock solution using analyte free urine to give the concentrations listed in Table 1. Calibration standards were then prepared using a dilution ratio strategy from the high concentration sample of 1:2:2:2.5:2. The high, medium and low QC samples were prepared using a dilution ratio strategy from the high concentration sample of 1:1.33:3.33: 8. b-Glucuronidase, Type-2, from Helix pomatia was purchased from Sigma-Aldrich. Fresh urine was obtained from a male volunteer. All other reagents and solvents used were reagent grade.
Instrumentation. All automated DPX PrepSequences were performed using a MultiPurpose Sampler (MPS XL Dual Tower) with GERSTEL DPX Option as shown in Figure 1. All analyses were performed using an Agilent® 1290 Infinity LC with a Zorbax Eclipse Plus C18 column (2.1 x 50 mm, 1.8 μm, 600 bar), an Agilent 6460 Triple Quadrupole Mass Spectrometer with Jet stream electrospray source and GERSTEL MPS XL autosampler configured with an Active Wash Station (AWS). Sample injections were made using a 6 port (0.25mm) Cheminert C2V injection valve fitted with a 2 μL stainless steel sample loop.
Sample pretreatment. Hydrolysis of urine was performed by combining 2 mL of urine, 150 μL of the working internal standard solution, 100 μL of b-Glucuronidase, and 500 μL of 0.66M acetate buffer, pH 4, vortex mixing for 30 seconds, and then incubating at 55°C for 2 hours. Aliquots of 260 μL of hydrolyzed urine samples were added into clean shell vials for automated cleanup and injection. The automated extraction (DPX Prep Sequence and Clean-up procedure) used for this method consisted of the following steps:

  1. Aspirate 750 μL of 100 % acetonitrile from the fast solvent delivery station using the 2.5 mL DPX syringe.
  2. Pick up a new DPX tip (DPX-RP-S) located within the tray.
  3. Add 500 μL of 100 % acetonitrile through the DPX tip, into the urine sample located on the MPS sample tray.
  4. Wait for 6 seconds to allow the acetonitrile to completely wet the DPX sorbent.
  5. Aspirate the entire sample followed by 1400 μL of air into the DPX tip.
  6. After equilibrating for 5 seconds, dispense the contents of the DPX tip, as well as the remaining acetonitrile found within the DPX syringe, back into the original shell vial in the tray.
  7. Move the DPX tip to the PipWaste position and dispose of the DPX tip.
  8. Transfer 100 μL of the upper liquid layer located within the original shell vial, into a clean, empty, capped autosampler vial with magnetic septum cap located on a VT98 tray.
  9. Dilute the extract by adding 900 μL of water into the sample vial.
  10. Inject 50 μL of the sample into the HPLC injection valve (2 μL injection loop).

A total of 116 MRM transitions (98 Analyte qualifier/quantifier and 18 internal standard transitions) were monitored in a 4 minute analytical window followed by a column regeneration time of 2.5 minutes. A retention time window of 30 seconds was used for each positive ion transition monitored in the dynamic MRM method. Detailed mass spectrometric acquisition parameters are available upon request.

RESULTS AND DISCUSSION

Figure 3 shows representative dynamic MRM chromatograms for all 49 pain management drugs and internal standards in a hydrolyzed urine sample spiked at the minimum reporting limit (MRL) and cleaned using automated DPX.
Table 1 lists the column retention times, concentrations for the highest calibration standard, MRLs and LLOQs for the 49 analytes in this screening assay. LLOQ concentrations are higher (5 fold factor increase) in comparison to those listed in our previous work performed with an automated concentration step using a solvent evaporation station [1]. However, the LLOQs of this modified cleanup method are still below the original MRLs and cycle times are considerably shorter. Representative calibration curves are shown in Figure 4. Regression analysis for all pain management drugs analyzed within this method resulted in R2 values of 0.99 or greater.
The DPX automated sample cleanup time was reduced from 7 to 3 min/sample; the total cycle time per sample for the extraction process and injection was reduced from 13 to 7 min/sample, fitting with the “just in time” sample preparation strategy available using the MAESTRO software and increasing throughput. Using this automated procedure for extraction and analysis over 200 samples can be processed per day. The accuracy and precision of the method was measured for all pain management drugs analyzed by extracting replicate QC samples (n=4) at high and low concentrations. Table 2 shows the resulting accuracy and precision data for all pain management drugs. Accuracy data averaged 98.0 % (range: 77 - 110 %) and precision data (% RSD) averaged 4.2 % (range: 0.9 -12.8 %) for all pain management drugs determined.

CONCLUSIONS

As a result of this study, we were able to show:

  • The automated DPX cleanup method using the GERSTEL MPS XL Dual Tower robotic sampler for pain management drug screenings in urine provided cycle times of approximately 7 min/sample allowing throughput of over 200 samples per day.
  • 49 pain management drugs can be rapidly and reproducibly isolated from hydrolyzed urine samples using an automated DPX cleanup procedure coupled to LCMS/ MS analysis using the Agilent 6460 Triple Quadrapole Mass Spectrometer.
  • Linear calibration curves resulting in R2 values 0.99 or greater were achieved with LOQs lower than the minimum reporting limits for the majority of pain management drugs analyzed.
  • The DPX-LC-MS/MS method provided good accuracy and precision averaging 98.0 % (range 77 - 110 %) accuracy with 4.2 % RSD (range: 0.9 -12.8 %) for all pain management drugs analyzed.
Compound Ret. Time
[min]
High Cal Std.
[ng/mL]
MRL
[ng/mL]
LOQ
[ng/mL]
6-MAM1 1.60 100 10 5
Codeine1 1.43 500 50 25
Hydrocodone1 1.56 500 50 25
Hydromorphone1 1.01 500 5 25
Oxycodone1 1.51 500 50 25
Morphine1 0.71 500 50 25
Oxymorphone1 0.83 500 50 25
Meperidine1 2.27 500 50 25
Normeperidine1 2.33 500 50 25
Buprenorphine2 3.02 100 10 5
Norbuprenorphine3 2.60 100 10 5
EDDP4 2.76 500 50 25
Methadone4 3.22 500 50 25
Norpropoxyphene5 2.98 1000 100 50
Propoxyphene5 3.16 1000 100 50
O-Desmethyl-cis-Tramadol6 1.71 25 25 12.5
cis-Tramadol6 2.13 250 25 12.5
Fentanyl7 2.64 10 1 0.5
Norfentanyl7 2.04 10 1 0.5
Meprobamate8 2.58 500 50 25
Carisoprodol8 3.38 500 50 25
7-aminoclonazepam9 2.12 400 40 20
Clonazepam10 3.17 400 40 20
Oxazepam11 3.38 400 40 20
Estazolam12 3.30 400 40 20
Alprazolam13 3.42 400 40 20
Diazepam13 3.75 400 40 20
Flunitrazepam13 3.23 400 40 20
Lorazepam12 3.39 400 40 20
Nitrazepam13 3.15 400 40 20
Temazepam13 3.50 400 40 20
α-OH-alprazolam14 3.29 400 40 20
Nordiazepam13 3.63 400 40 20
Bromazepam12 3.05 400 40 20
Clobazam13 3.34 400 40 20
Midazolam13 3.08 400 40 20
Triazolam13 3.41 400 40 20
Flurazepam13 2.79 400 40 20
Ketamine15 2.01 1000 100 50
Norketamine15 2.01 1000 100 50
Amphetamine16 1.60 1000 100 50
MDA16 1.64 1000 100 50
MDEA16 1.82 1000 100 50
MDMA16 1.69 1000 100 50
Methamphetamine16 1.66 1000 100 50
Methylphenidate16 2.16 1000 100 50
PCP17 2.54 50 5 2.5
Benzoylecgonine18 1.99 250 25 12.5
Cocaine18 2.13 250 25 12.5
Table 1. Retention times, high calibration standard concentrations, MRLs and LOQs for all pain management drugs analyzed. (1 - 18 used internal standard)
1) d3-morphine 6) d3-cistramadol 11) d5-oxazepam 16) d5-amphetamine
2) d4-buprenorphine 7) d5-Fentanyl 12) d5-estazolam 17) d5-PCP
3) d3-norbuprenorphine 8) d7-Carisoprodol 13) d5-nordiazepam 18) d3-cocaine
4) d9-methadone 9) d4-7-aminoclonazepam 14) d5-α-OH-alprazolam
5) d5-propoxyphene 10) d4-Clonazepam 15) d4-ketamine
List of internal standards used.
Compound QCL
[ng/mL]
Avg. Accuracy [%]
[n = 4]
QCH
%RSD
 
[ng/mL]
Avg. Accuracy [%]
[n = 4]
 
%RSD
6-MAM 12.5 101 12.7 75.0 107 4.9
Codeine 62.5 98 3.0 375.0 105 2.3
Hydrocodone 62.5 96 3.8 375.0 104 2.7
Hydromorphone 62.5 99 2.3 375.0 102 1.6
Oxycodone 62.5 98 2.9 375.0 103 2.8
Morphine 62.5 97 6.5 375.0 102 1.5
Oxymorphone 62.5 100 2.6 375.0 101 3.0
Meperidine 62.5 97 3.2 375.0 104 4.5
Normeperidine 62.5 98 2.6 375.0 103 4.0
Buprenorphine 12.5 103 10.8 75.0 106 4.2
Norbuprenorphine 12.5 91 9.3 75.0 97 7.7
EDDP 62.5 106 1.5 375.0 107 1.7
Methadone 62.5 107 3.0 375.0 108 2.2
Norpropoxyphene 125.0 96 1.9 300.0 97 1.5
Propoxyphene 125.0 96 2.2 300.0 97 1.0
o-Desmethyl-cis-Tramadol 31.3 94 2.1 187.5 93 1.3
Tramadol 31.3 94 2.6 187.5 94 0.9
Fentanyl 1.3 109 7.0 7.5 102 4.0
Norfentanyl 1.3 99 12.1 7.5 98 2.8
Meprobamate 62.5 87 3.3 375.0 88 1.1
Carisoprodol 62.5 90 2.8 375.0 84 1.1
7-aminoclonazepam 50.0 90 10.1 300.0 92 6.2
Clonazepam 50.0 89 9.1 300.0 101 2.6
Oxazepam 50.0 84 12.9 300.0 97 5.9
Estazolam 50.0 87 3.7 300.0 90 3.2
Alprazolam 50.0 100 3.3 300.0 104 5.5
Diazepam 50.0 98 3.8 300.0 98 4.5
Flunitrazepam 50.0 109 6.9 300.0 94 4.3
Lorazepam 50.0 92 6.7 300.0 97 8.7
Nitrazepam 50.0 97 12.6 300.0 97 5.0
Temazepam 50.0 95 7.8 300.0 98 2.2
α-OH-alprazolam 50.0 77 4.1 300.0 79. 6.2
Nordiazepam 50.0 110 10.6 300.0 96 5.8
Bromazepam 50.0 104 12.0 300.0 90 2.3
Clobazam 50.0 102 5.3 300.0 97 4.4
Midazolam 50.0 102 5.3 300.0 97 4.4
Triazolam 50.0 100 4.2 300.0 104 6.3
Flurazepam 50.0 101 3.2 300.0 98 4.2
Ketamine 125.0 88 1.4 300.0 89 1.0
Norketamine 125.0 91 2.1 300.0 88 1.4
Amphetamine 125.0 100 2.4 300.0 102 1.5
MDA 125.0 99 2.5 300.0 103 1.4
MDEA 125.0 102 2.5 300.0 103 1.7
MDMA 125.0 101 2.4 300.0 101. 1.4
Methamphetamine 125.0 101 2.6 300.0 103 1.4
Methylphenidate 125.0 99 2.3 300.0 103 2.0
PCP 6.3 107 7.3 37.5 105 3.0
Benzoylecgonine 31.3 97 3.4 187.5 98 2.5
Cocaine 31.25 98 3.3 187.5 100 1.2
Table 2. Extracted QC samples % accuracies and % RSDs.

References

[1] Determination of Pain Management Drugs using Automated Disposable Pipette Extraction and LC-MS/MS, GERSTEL AppNote AN-2011-06

 

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