Pierre Picard (Presenter)
Phytronix Technologies
Authorship: Pierre Picard1, Stephanie J. Marin2, Gwendolyn A. McMillin2,3, Alex Birsan1, Serge Auger1 and Jean Lacoursière1
1) Phytronix Technologies, Quebec, Canada 2) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 3) University of Utah, Salt Lake City, UT
| Short Abstract Drug abuse during pregnancy is a major medical issue associated with significant maternal and infant complications. Meconium is a common specimen used to identify drug-exposed infants. The proposed mechanism for drug presence in the meconium is that the fetus excretes the drug into bile and amniotic fluid. Current analysis method uses several immunoassays to cover many drugs. To reduce the number of screening reagents, required quantity of meconium and analysis time, a Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS) method was developed. A fast extraction method is used with calibration range of: 20 to 200 ng/g for amphetamines/cocaine/opiate/oxycodone/PCP/methadone and 50 to 500 ng/g for Barbiturates/Benzodiazepines classes. The lower limit of the calibration curves represents the cutoff for reporting results. Sample analysis time is 9 seconds per sample. |
Long Abstract
Overview
Drug abuse during pregnancy is a major medical issue associated with significant maternal and infant complications. Meconium is a common specimen used to identify and characterize drug-exposed infants. The proposed mechanism for drug presence in the meconium is that the fetus excretes the drug into bile and amniotic fluid. Drug accumulates in the meconium either by direct deposit from bile or through swallowing of the amniotic fluid. ARUP Laboratories uses immunoassay to screen for the presence of different drug families. To reduce the number of screening assays and reduce the quantity of meconium required for testing, a Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS) method was developed.
Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS) offers specificity combined with an ultra-fast analysis for an unrivaled screening method. A fast and simple extraction method is described, with the following calibration range: 20 to 200 ng/g of meconium for amphetamines/cocaine/opiate/oxycodone/PCP/methadone classes and 50 to 500 ng/g of meconium for Barbiturates/Benzodiazepines classes. The lower limit of the calibration curves served as the cutoff for reporting results.
LDTD Ionization Source
The LDTD uses a Laser Diode to produce and control heat on the sample support which is a 96 well plate. The energy is then transferred through the sample holder to the dry sample which vaporizes prior to being carried by a gas in an APCI region. High efficiency protonation with strong resistance to ionic suppression characterize the ionization due to the absence of solvent and mobile phase. This allows very high throughput capabilities of 9 seconds sample-to-sample analysis time, without any carry over.
Sample preparation
Meconium preparation procedure
• 0.1 g Meconium in 1.5 mL Eppendorf tube.
• 1 mL Phosphate buffer (0.1M, pH 7)
Vortex and sonic 20 minutes
Centrifuge 14000 rpm / 10 minutes
• Filtrate solution using 0.45µ filter
Meconium drug spiking procedure
• 10 µL working drug solution in Methanol
• 200 µL Meconium preparation solution
Vortex
Enzymatic hydrolysis
• 110 µL Meconium preparation in 0.5 mL Eppendorf tube
• 5 µL Internal Standard (IS) solution
• 20 µL purified Beta-glucuronidase enzyme (IMCSzyme, >50 kU/mL)
• 25 µL rapid hydrolysis buffer (IMCSzyme)
• Vortex
• Incubate 15 minutes at 55°C
Extraction Procedure (Basic drugs)
• In the Meconium preparation / IS / Enzyme tube
• Add 100 µL Sodium carbonate buffer (0.5M pH 10)
Mix
• 200 µL Ethyl Acetate
Mix
Centrifuge 14000 rpm/2min
• Transfer 4 µL of organic layer in LazWell plate*
• Dry prior to analysis
Extraction Procedure (Acidic drugs)
• In the Meconium preparation / IS / Enzyme tube
• Add 200 µL NaCl (saturate solution in water)
Mix
• 400 µL Acetonitrile
Mix
Centrifuge 14000 rpm/2min
• Transfer 4 µL of organic layer in LazWell plate (Phytronix) *
• Dry prior to analysis
*LazWell plate coating:
96-well plates for analysis are pre-coated with 5 µL of an EDTA solution (100 µg/mL in MeOH/H2O/NH4OH (75/20/5%)) and dried before sample deposition.
Instrument settings
1) LDTD:
Phytronix LDTD model S-960 operated with a gas flow rate of 3 L/min and a laser pattern ramp from 0 to 65% in 6 seconds.
2) Method for basic drugs (Positive ionization method):
Mass spectrometer Sciex 5500 QTrap
MRM transitions used with 3 µA corona discharge current, 5 msec dwell time, and a 100 V DP
Compounds Q1 Q3 CE(V)
Alprazolam 311 274 40
Diazepam 285 154 32
OH-Alprazolam 325 205 54
Oxazepam 287 241 30
Temazepam 301 255 25
D5-Oxazepam 292 246 32
D5-Temazepam 306 260 25
MDA 180 133 20
MDEA 208 163 12
MDMA 194 163 12
Amphetamine 136 119 15
Methamphetamine 150 119 15
D9-Methamphetamine 159 125 15
PCP 244 159 20
D5-PCP 249 164 20
Methadone 310 265 20
MOR-HYM 286 165 50
Oxycodone 316 241 40
Oxymorphone 302 227 40
COD-HYC 300 215 35
D9-Methadone 319 268 20
3) Method for acidic drugs (Positive/Negative ionization method):
Mass spectrometer Sciex 5500 QTrap
MRM transitions used with 3 µA corona discharge current, 5 msec dwell time, and a 100 V DP using positive and negative mode.
Positive:
Compounds Q1 Q3 CE(V)
BZE 290 168 25
BZE-d8 298 171 25
Negative:
Compounds Q1 Q3 CE(V)
Amobarbital 225 182 -15
Phenobarbital 231 42 -45
Secobarbital 237 42 -45
Butalbital 223 42 -45
Butabarbital 211 42 -45
D5-Phenobarbital 236 42 -45
Methodology
The standard calibration curve is made with a stock solution of each drug spiked in blank meconium matrix preparation. Standard concentrations for drug classes Amphetamine, PCP, Methadone, Opiates/Oxycodone and Cocaine are 20, 50, 100 and 200 ng/g. Standard concentrations for drug classes Benzodiazepine and Barbiturates are 50, 125, 200 and 500 ng/g. The ISs are prepared with a stock solution of each IS, spiked in methanol for a final concentration of 200 ng/mL. Each sample is hydrolyzed using a β-glucuronidase faster hydrolysis process (15 minutes) with purified enzyme. Specific LLE extraction procedures are performed for basic and salt assisted liquid-liquid extraction (SALLE) for acidic drugs. All samples are then analyzed using LDTD-MS/MS method.
Results
For each drug, peak area against internal standard signal ratio was used for signal normalization. The precision tests at the decision point were used to evaluate the analytical technique performance. The curves for each concentration showing the mean plus or minus two times the standard deviation for each sample must not overlap for the decision point to be valid.
According to the two times the standard deviation rule for the precision test, the overlap graphs are drawn. In figure 1, the curves for Methadone are showed.
Conclusion:
After the Meconium dissolution, the hydrolysis step using purified β-glucuronidase is used to obtain the free drug form in matrix. Two specific extraction procedures for basic and acidic drugs are then performed. The LDTD technology combined with a mass spectrometer system allows ultra-fast and specific drug screening in meconium matrix samples. One MRM method and one well are used to screen basic drug and another well is used for analysis of acid drugs at 9 seconds per sample.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Phytronix Technologies |
| Board Member | no | |
| Stock | yes | Phytronix Technologies |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |