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Abstract INTRODUCTION:
Free Opiates test is one of the high-volume tests at the NMS laboratory. This test covers wide range of biologic samples such as in whole blood, postmortem blood, serum/plasma, vitreous fluid and urine. Due to current method that is tedious, costing and time-consuming with high repeatable rate, the organization decided to redevelopment of the current test. The challenge of the redevelop the method is to achieve a faster and cost-effective method with extended analytical measurement ranges (AMR) to reduce the sample repeatable rates. The work described here is the new development of free opiates ( Morphine, Hydromorphone, Codeine, Dihydrocodeine, 6-MAM, Oxycodone and hydrocodone) in both sample preparation and chromatographic separations with AMRs extended up to 2.5 times and the final ranges are 1-200 ng/mL for 6-MAM, oxymorphone and hydromorphone; 5-500 ng/mL for codeine and dihydrocodeine; 5-1000 ng/mL for all other analytes in the panel.
METHODS:
A Bioatge Isolute SLE+ 400 µL plate on ExtraHera was used for sample preparation. After 200 µL of calibrators, QCs and samples are aliquoted on the SLE plate, 25 µL of internal standard was added to each well followed by 255 µL 1.5% NH4OH solution. The plate was then mixed and spin down at 4000 rpm for 10 minutes before load onto the Extrahera. A “load only” SLE method was applied to the automatic sample preparation. The Extrahera method will load 300 µL on to the extraction plate and elute three times with a total volume of 1350 µL of ethyl acetate. The receiving plate was drydown on a TurboVap-96 and reconstituted with 300 µL 95:5 (v:v) of mobile phase A : mobile phase B . Mobile phase A is 0.05% Formic acid with 5 mM Ammonium Acetate in water and mobile phase B is 0.05% formic acid in methanol.
The 96-well sample plate was mixed and spun at 4000 rpm for 5 min, then load onto a Waters Acquity UPLC/Xevo TQXS Triple Quadrupole Mass Spectrometer system. 2-µL sample was injected on LC-MSMS using a Phenomenex Kinetex Biphenyl column with dimensions of 50 x 2.1 mm, part. size 5 µm. The column temperature is maintained at 55 oC. The LC gradient starts at 2 % mobile phase B ( 0.1% formic acid in methanol) after hold for 0.6 min, mobile phase B was linearly gradient to 15% at 1.1 min; 20% at 1.9 min and 35% at 2.4 min respectively. From time 2.5-3.0 min, mobile phase B at 98% to wash the column and ready for the next injection.
RESULTS:
Under the chromatographic conditions, all the free opiates on the panel yield excellent peak shape and isobaric separations with a total analysis time of 3 minutes which is about 50% faster than previous method. The isobaric separations of morphine vs hydromorphone and codeine vs hydrocodone were achieved through the slow gradient at beginning and the oven temperature at 55 oC . The five intra-days and inter-days CVs are < 10% for all analytes in blood, serum and urine matrices. Interferences check with around 90 analytes that includes from broad OTCs, designer’s/synthetic opiates, cocaine panel, fentanyl panel, benzodiazepines, amphetamines and cannabinoids were performed during the new method validation. There were no interferences found under the method LC-MSMS conditions.
CONCLUSIONS:
A new SLE-LCMSMS method was developed, validated and now in live at the NMS laboratory. The method is accurate, sensitive and rugged. Compared to previous method, it is simper, faster and more cost effective. Not only the lower limits of quantitation were met, but the extended AMRs up to 2.5 times) were achieved with linear correlation coefficient > 0.997 for each analyte compared previous method. The extended AMRs reduced the sample repeatable rate significantly, as well as turnaround time. The method is adaptable for accurate and reproducible quantification of free opiates in variety of biologic matrices with high throughput at any laboratory.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
