For Research Use Only. NOT for Use in Diagnostic Procedures.
This application note describes a procedure for sample pre-treatment and extraction of a panel of 49 drugs of abuse from human hair, using Biotage® Lysera for matrix micropulverization, prior to clean up using ISOLUTE® SLE supported liquid extraction. The application note contains procedures optimized for 400 µL capacity column format and 200 µL and 400 µL supported liquid extraction plate formats, for higher throughput applications. The methodology delivers clean extracts and analyte recoveries mostly greater than 80% with RSDs lower than 10% for all analytes and LLOQ from 1 ng/mL.
ISOLUTE® SLE+ Supported Liquid Extraction plates and cartridges offer an efficient alternative to tradition liquid- liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced preparation time.
Amphetamine, Methamphetamine, MDA, MDMA, MDEA, Hydromorphone, Morphine, BZE, Oxymorphone, Dihydrocodeine, Oxycodone, Mephedrone, Norfentanyl, 7-amino-flunitrazepam, 7-amino-clonazepam, Hydrocodone, Codeine, 6-MAM, Cocaine, Norketamine, EDDP, Zaleplon, Zopiclone, Norbuprenorphine, Ketamine, Nitrazepam, Flunitrazepam, Clonazepam, a-OH-triazolam, Oxazepam, Estazolam, Temazepam, Zolpidem, Alprazolam, Methadone, Lorazepam, Bromazepam, a-OH-alprazolam, 2-OH-ethyl- flurazepam, Triazolam, Nordiazepam, Diazepam, Midazolam, Fentanyl, Flurazepam, Buprenorphine, PCP, LSD.
Amphetamine-D5, Morphine-D3, BZE-D3, 6-MAM-D3, Diazepam-D5
ISOLUTE® SLE+ 200 µL capacity plates (p/n 820-0200-P01)
ISOLUTE® SLE+ 400 µL capacity plates (p/n 820-0400-P01)
ISOLUTE® SLE+ 400 µL capacity cartridges (p/n 820-0055-B)
Weigh 20 mg of hair into 2 mL Biotage® Lysera tubes containing 4 x 2.4 mm stainless steel beads. Add 1 mL methanol to each hair sample spiked with ISTD (1 ng/mL).
Biotage® Lysera: 3 x 5.3 m/sec for 3 minutes with a 20s dwell.
Centrifuge tubes for 10 minutes at 13,300 rpm.
Transfer and evaporate extracts using a TurboVap® LV at 20 °C Reconstitute in up to 400 µL ( 50:50, v/v) methanol: water.
|
ISOLUTE SLE+ 200 µL Plate, |
ISOLUTE SLE+ 400 µL Plate, |
ISOLUTE SLE+ 400 µL cartridges, |
|
|
Part Number 820-0200-P01 |
Part Number 820-0400-P01 |
Part Number 820-0055-B |
|
|
Sample loading |
Load 200 µL of reconstituted supernatant to ISOLUTE® SLE+ sorbent and apply a pulse of pressure 3–5 seconds to initiate flow. Allow the sample to absorb for 5 minutes. |
Load 400 µL of reconstituted supernatant to ISOLUTE® SLE+ sorbent and apply a pulse of pressure 3–5 seconds to initiate flow. Allow the sample to absorb for 5 minutes. |
Load 400 µL of reconstituted supernatant to ISOLUTE® SLE+ sorbent and apply a pulse of pressure 3–5 seconds to initiate flow. Allow the sample to absorb for 5 minutes. |
|
Analyte Extraction |
Apply DCM/IPA (95/5, v /v, 300 µL) allow to flow under gravity for 5 minutes Apply a further aliquot of MTBE (300 µL) and allow to flow under gravity for 5 minutes. For complete removal apply a pulse of positive pressure at 10 psi (10–20 seconds). |
Apply DCM/IPA (95/5, v/v, 600 µL) allow to flow under gravity for 5 minutes. Apply a further aliquot of MTBE (600 µL) and allow to flow under gravity for 5 minutes. For complete removal apply a pulse of positive pressure at 10 psi (10–20 seconds). |
Apply DCM/IPA (95/5, v/v, 600 µL) allow to flow under gravity for 5 minutes. Apply a further aliquot of MTBE (600 µL) and allow to flow under gravity for 5 minutes. For complete removal apply a pulse of positive pressure at 10 psi (10–20 seconds). |
Evaporate extracts at 40 °C in the presence of 100 µL of 50 mM HCl in MeOH in order to avoid evaporative losses of amphetamines. For plate format, we used the Biotage® SPE Dry-96, and for column format we used the TurboVap® LV. Reconsitute using a mix of mobile phase A and mobile phase B (80/20, v/v, 200 uL).
Shimadzu Nexera UHPLC
Restek Raptor™ Biphenyl 2.7 µm (100 x 2.1 mm)
A: 2 mM Ammonium Formate (aq) 0.1% formic acid
B: 2 mM Ammonium Formate (MeOH) 0.1% formic acid
0.4 mL/min
5 μL
30 °C
Table 1. UHPLC gradient.
|
Time (min) |
%A |
%B |
|
0 |
80 |
20 |
|
2.00 |
80 |
20 |
|
7.50 |
40 |
60 |
|
11.25 |
40 |
60 |
|
12.75 |
0 |
100 |
|
13.50 |
0 |
100 |
|
13.51 |
80 |
20 |
|
15.00 |
80 |
20 |
Shimadzu 8060 Triple Quadrupole MS using ES interface
3 L/min
3 L/min
17 L/min
400 °C
250 °C
300 °C
270 kPa
Table 2. MS conditions for target analytes in positive mode.
|
Analytes |
MRM Transition |
Collision Energy |
Analytes |
MRM Transition |
Collision Energy |
|
Morphine-D3 |
289.0>201.1 289.0>152.1 |
-26.0 -50.0 |
LSD |
323.50>208.10 323.50>223.25 |
-29.0 -23.0 |
|
Morphine |
286.0>152.1 286.0>201.1 |
-50.0 -25.0 |
7-Aminoflunitrazepam |
283.90>135.05 283.90>227.05 |
-30.0 -26.0 |
|
Oxymorphone |
302.00>227.1 302.00>198.1 |
-30.0 -45.0 |
Zolpidem |
308.00>235.10 308.00>263.10 |
-35.0 -25.0 |
|
Hydromorphone |
286.0>185.0 286.0>157.0 |
-30.0 -40.0 |
Buprenorphine |
468.10>396.25 468.10>414.30 |
-40.0 -35.0 |
|
Amphetamine-D5 |
141.0>93.0 141.0>124.15 |
-15.0 -20.0 |
Fentanyl |
337.00>188.10 337.00>105.00 |
-20.0 -40.0 |
|
Amphetamine |
136>91.05 136>119.1 |
-15.0 -14.0 |
Flurazepam |
388.00>315.00 388.00>288.00 |
-20.0 -26.0 |
|
Methamphetamine |
150.0>90.95 150>119.1 |
-20.0 -14.0 |
PCP |
244.00>91.05 244.00>159.15 |
-35.0 -14.0 |
|
MDA |
180>105 180>77 |
-20.0 -40.0 |
Midazolam |
325.90>249.10 325.90>223.00 |
-35.0 -40.0 |
|
Dihydrocodeine |
302>119.05 302>171 |
-35.0 -45.0 |
Bromazepam |
315.80>182.10 315.80>209.10 |
-31.0 -27.0 |
|
Codeine |
300.0>215.1 300.0>165 |
-25.0 -40.0 |
EDDP |
278.00>234.00 278.00>234.00 |
-30.0 -45.0 |
|
6-MAM-D3 |
331.0>165.1 331.0>211.1 |
-40.0 -25.0 |
Lorazepam |
320.80>275.00 320.80>229.05 |
-22.0 -30.0 |
|
6-MAM |
328.0>165.1 328.0>211.1 |
-40.0 -25.0 |
Oxazepam |
320.80>229.05 286.90>104.20 |
-23.0 -35.0 |
|
MDMA |
194.0>163.1 194.0>105.0 |
-15.0 -25.0 |
Nitrazepam |
286.90>104.20 281.90>180.10 |
-25.0 -35.0 |
|
Oxycodone |
316.2>241.2 |
-20.0 |
Clonazepam |
315.90>270.05 315.90>214.05 |
-25.0 -38.0 |
|
Mephedrone |
178.00>145.05 178.00>144.00 |
-20.0 -30.0 |
a-OH-Triazolam |
358.90>331.10 358.90>239.05 |
-28.0 -44.0 |
|
Hydrocodone |
300.0>199.05 300.0>171.1 |
-30.0 -40.0 |
2-OH-et-flurazepam |
332.90>211.10 332.90>109.00 |
-37.0 -27.0 |
|
MDEA |
208>163.05 208>105.05 |
-15.0 -25.0 |
Methadrone |
310.50>265.10 |
-16.0 |
|
Nor-Ketamine |
223.9>125 223.9>179.05 |
-20.0 -15.0 |
a-OH-Alprazolam |
324.90>216.10 324.90>205.10 |
-39.0 -46.0 |
|
Nor-Fentanyl |
233.0>84.05 233.0>56.05 |
-20.0 -26.0 |
Nordiazepam |
270.90>140.05 270.90>208.10 |
-26.0 -28.0 |
|
BZE-D3 |
293.00>171.05 293.00>77.00 |
-20.0 -50.0 |
Zaleplon |
305.90>236.15 305.90>264.20 |
-28.0 -22.0 |
|
BZE |
289.90>168.05 289.90>105.00 |
-20.0 -30.0 |
Flunitrazepam |
313.90>268.10 313.90>239.10 |
-25.0 -35.0 |
|
Ketamine |
237.90>125.00 237.90>207.05 |
-30.0 -14.0 |
Estazolam |
294.90>267.05 294.90>205.05 |
-20.0 -40.0 |
|
7-Aminoclonazepam |
285.90>222.10 285.90>121.10 |
-25.0 -29.0 |
Temazepam |
300.90>255.05 300.90>177.05 |
-20.0 -39.0 |
|
Cocaine |
304.00>182.05 304.00>82.05 |
-20.0 -30.0 |
Triazolam |
342.90>308.10 342.90>239.05 |
-27.0 -41.0 |
|
Zopiclone |
388.90>245.05 388.90>217.00 |
-15.0 -35.0 |
Alprazolam |
308.90>281.00 308.90>205.05 |
-25.0 -40.0 |
|
Norbuprenorphine |
414.00>101.25 414.00>187.20 |
-39.0 -38.0 |
Diazepam-D5 |
289.90>193.05 289.90>258.00 |
-32 -7.0 |
|
|
|
|
Diazepam |
285.10>193.05 285.10 |
-32.0 -27.0 |
This simple sample preparation method delivers clean extracts and analyte recoveries mostly greater than 80% with RSDs lower than 10% for all analytes (see Figure 2), and LLOQs from 1 ng/mL (see table 3) for all ISOLUTE® SLE+ formats used.
Figure 2. Representative analyte recoveries using the optimized SLE protocol for the 400 µL capacity column format (p/n 820-0055-B. Similar results were achieved using the 200 µL and 400 µL capacity plate formats.
Figure 3. Representative chromatography for application analytes spiked at 1 ng/mL.
Calibration curve performance was investigated from hair (analytes (1–1000 pg/mL) were added to methanolic NH4OH extraction solvent prior to micropulverization) Good linearity was observed for all analytes typically delivering r2 values greater than 0.99. Table 3. details linearity performance and associated LOQ for each analyte, using the 400 µL capacity column format, p/n 820-0055-B. Similar results were achieved using the 200 µL and 400 µL capacity plate formats.
Figure 4. Calibration curves for Burprenorphine (a), 6-MAM (b), BZE (c) and Methamphetamine (d) extracted from human hair using the 400 µL capacity column format loading 400 µL of reconstituted extract. Similar results were achieved for the 200 µL and 400 µL capacity plate formats.
Table 3. Analyte calibration curve r2 and LOQ performance.
|
Analyte |
400 µL Load r2 |
400 µL Load LLOQ (pg/mL) |
|
Morphine |
0.998 |
1 |
|
Oxymorphone |
0.998 |
1 |
|
Hydromorphone |
0.997 |
1 |
|
Amphetamine |
0.998 |
1 |
|
Methamphetamine |
0.998 |
1 |
|
MDA |
0.998 |
10 |
|
Dihydrocodeine |
0.998 |
<1 |
|
Codeine |
0.998 |
1 |
|
6-MAM |
0.999 |
<1 |
|
MDMA |
0.998 |
<1 |
|
Oxycodone |
0.998 |
1 |
|
Mephedrone |
0.996 |
1 |
|
Hydrocodone |
0.998 |
1 |
|
MDEA |
0.998 |
<1 |
|
Nor-Ketamine |
0.996 |
1 |
|
Nor-Fentanyl |
0.998 |
<1 |
|
BZE |
0.999 |
<1 |
|
Ketamine |
0.998 |
<1 |
|
7-Aminoclonazepam |
0.998 |
1 |
|
Cocaine |
0.997 |
<1 |
|
Zopiclone |
0.998 |
5 |
|
Norbuprenorphine |
0.998 |
50 |
|
LSD |
0.998 |
1 |
|
7-Aminoflunitrazepam |
0.999 |
1 |
|
Zolpidem |
0.998 |
<1 |
|
Buprenorphine |
0.996 |
1 |
|
Fentanyl |
0.998 |
<1 |
|
Flurazepam |
0.998 |
<1 |
|
PCP |
0.998 |
1 |
|
Midazolam |
0.997 |
1 |
|
Bromazepam |
0.998 |
5 |
|
EDDP |
0.999 |
<1 |
|
Lorazepam |
0.998 |
10 |
|
Oxazepam |
0.999 |
1 |
|
Nitrazepam |
0.999 |
5 |
|
Clonazepam |
0.998 |
5 |
|
a-OH-Triazolam |
0.997 |
5 |
|
2-OH-et-flurazepam |
0.998 |
10 |
|
Methadrone |
0.998 |
5 |
|
a-OH-Alprazolam |
0.995 |
10 |
|
Nordiazepam |
0.996 |
5 |
|
Zaleplon |
0.997 |
1 |
|
Flunitrazepam |
0.998 |
5 |
|
Estazolam |
0.998 |
<1 |
|
Temazepam |
0.998 |
5 |
|
Triazolam |
0.998 |
1 |
|
Alprazolam |
0.997 |
<1 |
|
Diazepam |
0.997 |
1 |
|
Part Number |
Description |
Quantity |
|
19-060 |
Biotage® Lysera |
1 |
|
19-649 |
Bulk 2 mL Reinforced Tubes with Screw Caps |
1000 |
|
19-640 |
Bulk 2.4 mm Metal Beads – 500 g |
1 |
|
820-0055-B |
ISOLUTE® SLE+ 400 µL Sample Volume Cartridges |
50 |
|
820-0200-P01 |
ISOLUTE® SLE+ 200 µL Capacity Plate |
1 |
|
820-0400-P01 |
ISOLUTE® SLE+ 400 µL Capacity Plate |
1 |
|
PPM-96 |
Biotage® PRESSURE+ 96 Positive Pressure Manifold |
1 |
|
415000 |
TurboVap® LV |
1 |
|
SD-9600-DHS |
Biotage® SPE Dry Sample Concentrator System |
1 |
Literature Number: AN898