Electrospray LC-MS Method with Solid-Phase Extraction for Accurate Determination of Morphine-, Codeine-, and Ethylmorphine-Glucuronides and 6-Acetylmorphine in Urine

Abstract

A method for the identification and quantification of morphine-3-glucuronide, codeine-6-glucuronide, ethylmorphine-6-glucuronide, and 6-acetylmorphine in human urine based on solid-phase extraction (SPE) and electrospray ionization liquid chromatography-mass spectrometry (LC-MS) was validated for use as a confirmation procedure in combination with immunochemical screening for opiates. Three deuterium-labelled analogues were used as internal standards: morphine-3-glucuronide-d3, codeine-d3, and 6-acetylmorphine-d3. Fifty-microliter aliquots of urine were prepared by SPE using 30-mg Oasis HLB cartridges. The chromatographic system consisted of a 2.0 x 100-mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. The protonated molecular ions were monitored in the selected ion monitoring mode together with one qualifier ion for each analyte. The interassay variability was less than 10% at the reporting limit 30 ng/mL for 6-acetylmorphine and 300 ng/mL for the other analytes. The method was validated by comparison with a reference gas chromatographic (GC)-MS method using authentic urine samples. The two methods agreed completely regarding identified analytes, and for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by GC-MS. This result was to be expected because the free compounds are not measured with the LC-MS method. This study concludes that the presented LC-MS method is robust and reliable, and suitable for use as a confirmation method in clinical urine drug testing for opiates.

On-line solid phase extraction coupled with liquid chromatography tandem mass spectrometry for the determination of codeine in human plasma

“…This method awards compensation over those previously reported [6][7][8][9][10][11][12][13]23 in terms of time per sample clean-up, improved separation speed by monolithic column and high-throughput due to fast chromatographic run time synchronized to sample extraction (3.0 min). On-line SPE-LC-MS/MS method allowed a rapid assay development and sample processing.…”

“…Inter-day accuracy ranged from 0.03 to 3.80%, and inter-day precision ranged from 4.35 to 7.25, as shown in Table 2. Although, the toxicological and forensic methods [6][7][8][9][10][11][12][13] described here are sufficiently precise and accurate for the measurement of codeine in human plasma, this current method showed excellent results, with intra and inter-day accuracy and precision less than 10% and within the ranges specified by ANVISA guideline. 17 Other automated and pharmacokinetics determination of the codeine 18-23 also failed to achieve intra-day precision less than 5% as was obtained in this study.…”

“…Monolithic columns improve high-throughput due to higher separation efficiency at high flow velocities when compared to conventional LC columns. 16 Svensson et al 9 determined codeine retention time of approximately 5 min using solid phase extraction (SPE) and conventional C18 column chromatography. Most forensic application methods determined codeine with long chromatographic run time.…”

“…Most forensic application methods determined codeine with long chromatographic run time. [6][7][8][9][10][11][12][13] Therefore, mobile phase optimization is important to improve peak shape and sensitivity detection as well as codeine and tramadol chromatographic run time reduction (IS).…”

“…Several studies have demonstrated that it is still a drug of choice for treating acute and chronic pain. [1][2][3][4][5] Although, many bioanalytical methods have been developed for determination of codeine by LC-MS/MS and CG-MS, such as toxicological and forensic aplicattions, [6][7][8][9][10][11][12][13] most of these methods are based on off-line liquid-liquid or solid phase extraction (SPE). These procedures require complex steps and laborious repetitive work for sample clean-up, which may not meet the requirement for high-throughput, robustness and speed in bioanalysis and pharmacokinetic studies.…”