Evaluation of Drugs of Abuse Extraction from Oral Fluid using Supported Liquid Extraction (SLE) prior to UPLC-MS/MS Analysis

Lee Williams1, Rhys Jones1, Helen Lodder1, Geoff Davies1, Adam Senior1, Alan Edgington1, Steve Jordan1, Claire Desbrow1, Victor Vandell2, Frank Kero2. 1Biotage GB Limited, Distribution Way, Dyffryn Business Park, Ystrad Mynach, Cardiff, CF82 7TS, UK. 2Biotage, 10430 Harris Oaks Blvd., Suite C, Charlotte North Carolina 28269, USA.

© 2014 Biotage. All rights reserved. All trademarks are the property of their respective companies. www.biotage.com Part Number: P112

Introduction Drug screening using oral fluid has gained popularity over recent years due to its simple, non-invasive collection means. Screening drugs of abuse can be complicated due to the wide variation of functional groups associated with different analyte classes. Most extraction techniques cannot extract all analytes using a single procedure without using non-optimal extraction protocols, resulting in compromised extract cleanliness. Supported liquid extraction allows for the simultaneous analysis of cross functional analytes in a single extraction protocol without forfeiting extract cleanliness. This poster demonstrates the extraction of a range of drugs of abuse prior to UPLC-MS/MS analysis. The target analyte list includes benzodiazepines, z drugs, amphetamines, cathinones, opiates, cocaine, buprenorphine, PCP, THC-COOH, fentanyl and ketamine.

Experimental Reagents Drug standards were purchased from LGC Standards (Teddington, UK). Ammonium acetate, ammonium hydroxide (28-32%), HCl, formic acid were purchased from Sigma-Aldrich (Dorset, UK). Oral fluid collection devices were purchased from their respective companies. All solvents were HPLC grade from Fisher Scientific (Loughborough, UK) and Milli-Q (Merck Millipore, Germany) water used throughout.  Sample Preparation ISOLUTE® SLE+ Procedure (Figure 1.) Columns: ISOLUTE® SLE+ 400 µL capacity ‘B’ columns; 820-0055-B. ISOLUTE® SLE+ 1 mL capacity ‘C’ columns; 820-0140-C.

Matrix Pre-treatment: Intercept®: Add 10 µL of 0.5% NH4OH (v/v, aq) per device. Quantisal™: Add 15 µL of conc. NH4OH (aq) per device.

Sample Application: Quantisal™: 200*-400 µL (1:3 ratio of OF:buffer) was applied to the ISOLUTE SLE+ B column, or 500* µL (equivalent to 125 µL of OF) was applied to the ISOLUTE SLE+ C column. * maximum load for DCM combinations. Intercept®: 300 µL (1:2 ratio of OF:buffer) was applied to the ISOLUTE SLE+ B column, 300*-600 µL was applied to the ISOLUTE SLE+ C column. * maximum load for DCM combinations. Figure 1. Schematic of ISOLUTE® SLE+ Supported Liquid Scale up optimization using the Extraction Procedure.

Analyte Extraction: Quantisal™ and Intercept®: ISOLUTE SLE+ B columns: 2 x 1 mL aliquots of DCM. ISOLUTE SLE+ C columns: 2 x 3 mL aliquots of DCM. Each aliquot was allowed to flow under gravity for 5 minutes before applying a pulse of vacuum for 10-20 seconds to completely remove the final aliquot.

Post Extraction: 100 μL of 50 mM HCl in methanol was added to the collection tubes. The extracts were evaporated to dryness at 40 °C and reconstituted in 200 µL of 80:20 mobile phase prior to analysis.

UPLC-MS/MS Conditions UPLC: Waters Acquity UPLC (Waters Assoc., Milford, USA). Column: ACE EXCEL 1.7 µm C18 prototype column (100 x 2.1 mm id) (Advanced Chromatography Technologies, Aberdeen, UK). Mobile Phase: 5 mM NH4OAc (aq) and 5 mM NH4OAc/MeOH at a flow rate of 0.3 mL/min. Gradient: 90/10 increasing to 10/90 over 10 minutes. Initial starting conditions resumed at 11.4 minutes. Injection Volume: 10 µL (partial loop with overfill). Column Temperature: 40 ˚C. MS: Quattro Premier XE triple quadrupole mass spectrometer (Waters Assoc., Manchester, UK) equipped with an electrospray interface for mass analysis. Positive and negative ions were acquired in the MRM mode. Desolvation Temperature: 450 ˚C. Ion Source Temperature: 150 ˚C. Collision Gas Pressure: 3.5 x 10-3 mbar.

Results Each oral fluid collection device is made up of different combinations of additives to desorb the drugs from the collection pad and preserve the oral fluid matrix. Control of pH was investigated using minimal volumes of NH4OH to enable maximum matrix loading of the pre-buffered oral fluid. Loading pH was optimized to be between 8-8.5 to allow extraction of basic drugs while eliminating the conversion of 6-MAM to morphine. Additionally the extraction of native oral fluid from these devices demonstrated substantial residue and non-optimal cleanliness, so pH control was a necessity. Figures 2 and 3. demonstrate extraction recoveries obtained with various extraction solvents from the Quantisal™ and Intercept® collection devices, respectively. The cocaine metabolite, BZE required DCM combinations as the extraction solvent, preferentially 95/5 DCM/IPA. MTBE as a non-chlorinated solvent showed good recoveries of the majority of analytes investigated.

Figure 2. DOA recoveries from Quantisal™ oral fluid collection device.

Figure 3. DOA recoveries from Intercept® oral fluid collection device. The oral fluid collection device additives provide an interesting challenge when using LC-MS analysis. When performing full scans on the extracts it was noticed that substantial interference was observed when using DCM and 95/5 DCM/IPA as the extraction solvents. MTBE and EtOAc demonstrated less interference as shown in Figures 4 and 5. for Quantisal™ and Intercept® collection devices, respectively. For both collection devices the use of 95/5 DCM/IPA was discontinued. Optimization of loading volume provided the optimal balance of extract cleanliness and recoveries when using DCM.

Figure 4. Full scan TIC when using the Quantisal™ oral fluid collection device.

Figure 5. Full scan TIC when using the Intercept® oral fluid collection device. Figure 6. demonstrates analyte recoveries when using the Quantisal™ device with optimized loading volumes (200 µL

and 500 µL for the ISOLUTE 400 µL and 1 mL columns, respectively) and extraction with DCM.

Figure 6. Full scan TIC when using the Quantisal™ oral fluid collection device.

Figure 7. demonstrates typical calibration curves from 1-500 ng/mL obtained using the Quantisal™ device. Quadratic function was observed at high concentrations for a number of analytes. However, dilution and internal standards helped and ultimately demonstrated excellent linearity and coefficients of determination (r2 > 0.99). RSDs were below 10% at all concentration levels.

Figure 7. Calibration curves extracting 200 µL of Oral fluid using the Quantisal collection device urine using the SLE+ 400 µL columns.

Conclusion This poster demonstrates the suitability of ISOLUTE®

SLE+ for the rapid and reliable extraction of multiple drugs of abuse and metabolites from oral fluid.

BZE extraction requires the use of DCM. If BZE is not in the suite the use of other non-chlorinated solvents, such as MTBE can be substituted.

The use of 95/5 DCM/IPA although providing good extraction of most drug suites, suffers from non-optimal extract cleanliness associated with co-extraction of OF buffer additives.

Further Work Scale up optimization to ISOLUTE SLE+ C using the

Intercept® oral fluid collection device. Calibration lines using the Intercept® oral fluid

collection device.

Compound name: PCPCorrelation coefficient: r = 0.995387, r^2 = 0.990795Calibration curve: 1274.23 * x + 206.826Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 50 100 150 200 250 300 350 400 450 500

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pons

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Compound name: DiazepamCorrelation coefficient: r = 0.999326, r^2 = 0.998652Calibration curve: 1.40397 * x + 0.245954Response type: Internal Std ( Ref 48 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

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Compound name: AmphetamineCorrelation coefficient: r = 0.998077, r^2 = 0.996158Calibration curve: 0.810917 * x + 0.51846Response type: Internal Std ( Ref 7 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

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Compound name: BZECorrelation coefficient: r = 0.999439, r^2 = 0.998878Calibration curve: 0.887133 * x + 0.294319Response type: Internal Std ( Ref 11 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

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Time2.50 5.00 7.50 10.00

%

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100Scan ES+

TIC2.87e9

11.389.12

8.68

8.46

8.32

11.26

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC2.75e9

2.65

2.64

2.43

2.09

1.67

0.86

2.69

2.72

11.6211.37

2.7411.65

11.93

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC2.01e10

10.89

6.736.51

5.78

5.62

5.46

5.275.07

2.672.64

2.43

4.844.30

6.89

6.95

7.02

7.08

8.40

11.02

11.14

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC1.56e10

10.88

7.016.956.80

6.65

6.57

6.40

5.78

5.63

2.652.45

5.45

7.14

7.19

11.02

11.14

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC3.41e9

2.67

2.64

2.45

2.43

2.09

2.07

2.06

2.70

11.602.72

11.38

11.262.74

11.62

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC4.61e9

2.652.64

2.62

2.43

2.07

2.06

1.67

0.92

2.69

2.70

2.72

2.7410.97

2.75

2.77

2.79

7.79

4.302.81

4.324.37

9.349.17

11.01

Blank Solvent Injection 500 µL Load: DCM Extraction 1 mL Load: DCM Extraction 500 µL Load: 95/5 DCM/IPA Extraction 500 µL Load: MTBE Extraction 500 µL Load: EtOAc Extraction

Blank Solvent Injection 600 µL Load: DCM Extraction

Time2.00 4.00 6.00 8.00 10.00 12.00

%0

100Scan ES+

TIC2.27e10

11.16

10.43

5.555.35

4.94

4.714.58

4.29

4.15

3.973.80

3.470.85

5.69 9.48

5.81

5.95

6.12

6.256.36

6.46

6.57

11.87

Time2.00 4.00 6.00 8.00 10.00 12.00

%

0

100Scan ES+

TIC3.54e9

11.31

10.99

7.257.13

7.066.996.91

6.75

6.575.65

7.3510.29

7.86 9.83

8.64

11.89

Time2.00 4.00 6.00 8.00 10.00 12.00

%

0

100Scan ES+

TIC6.19e9

11.31

11.25

10.41

7.307.11

0.853.49

2.702.21

6.98

6.67

4.52

9.43

7.79

11.89

300 µL Load: DCM Extraction

Time2.50 5.00 7.50 10.00

%

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100Scan ES+

TIC2.31e10

11.27

10.55

10.48

9.56

600 µL Load: MTBE Extraction

Time2.50 5.00 7.50 10.00

%

0

100Scan ES+

TIC2.31e10

11.26

11.04

10.55

9.56

11.70

600 µL Load: EtOAc Extraction

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Format Scale Up Loading From Quantisal

B Column Format C Column Format

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Extraction Solvent Screen: Intercept Oral Fluid Device

MTBE DCM 95/5 DCM/IPA

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Extraction Solvent Screen: Quantisal Oral Fluid Device

MTBE 95/5 DCM/IPA