Functional phenotyping of the CYP2D6 probe drug codeine in the horse.

BACKGROUND: In humans, the drug metabolizing enzyme CYP2D6 is highly polymorphic resulting in substantial differences in the metabolism of drugs including anti-arrhythmics, neuroleptics, and opioids. The objective of this study was to phenotype a population of 100 horses from five different breeds and assess differences in the metabolic activity of the equine CYP2D6 homolog using codeine as a probe drug. Administration of a probe drug is a common method used for patient phenotyping in human medicine, whereby the ratio of parent drug to metabolite (metabolic ratio, MR) can be used to compare relative enzyme function between individuals. A single oral dose of codeine (0.6 mg/kg) was administered and plasma concentrations of codeine and its metabolites were determined using liquid chromatography mass spectrometry. The MR of codeine O-demethylation [(codeine)/(morphine + morphine-3-glucuronide + morphine-6-glucuronide)] was determined using the area under the plasma concentration-time curve extrapolated from time zero to infinity (AUC0-∞) for each analyte and used to group horses into predicted phenotypes (high-, moderate-, and low-MR). RESULTS: The MR of codeine O-demethylation ranged from 0.002 to 0.147 (median 0.018) among all horses. No significant difference in MR was observed between breeds, age, or sex. Of the 100 horses, 11 were classified as high-MR, 72 moderate-MR, and 17 low-MR. Codeine AUC0-∞ and O-demethylation MR were significantly different (p < 0.05) between all three groups. The mean ± SD MR was 0.089 ± 0.027, 0.022 ± 0.011, and 0.0095 ± 0.001 for high-, moderate-, and low-MR groups, respectively. The AUC for the morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were significantly different between high-and low-MR groups (p < 0.004 and p < 0.006). CONCLUSIONS: The MR calculated from plasma following codeine administration allowed for classification of horses into metabolic phenotypes within a large population. The range of codeine metabolism observed among horses suggests the presence of genetic polymorphisms in CYP2D82 of which codeine is a known substrate. Additional studies including CYP2D82 genotyping of high- and low-MR individuals are necessary to determine the presence of CYP2D polymorphisms and their functional implications with respect to the metabolism of therapeutics.

Metabolism, pharmacokinetics and selected pharmacodynamic effects of codeine following a single oral administration to horses.

OBJECTIVE: To describe the pharmacokinetics and selected pharmacodynamic variables of codeine and its metabolites in Thoroughbred horses following a single oral administration. STUDY DESIGN: Prospective experimental study. ANIMALS: A total of 12 Thoroughbred horses, nine geldings and three mares, aged 4-8 years. METHODS: Horses were administered codeine (0.6 mg kg-1) orally and blood was collected before administration and at various times until 120 hours post administration. Plasma and urine samples were collected and analyzed for codeine and its metabolites by liquid chromatography-mass spectrometry, and plasma pharmacokinetics were determined. Heart rate and rhythm, step counts, packed cell volume and total plasma protein were measured before and 4 hours after administration. RESULTS: Codeine was rapidly converted to the metabolites norcodeine, codeine-6-glucuronide (C6G), morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Plasma codeine concentrations were best represented using a two-compartment model. The Cmax, tmax and elimination t½ were 270.7 ± 136.0 ng mL-1, 0.438 ± 0.156 hours and 2.00 ± 0.534 hours, respectively. M3G was the main metabolite detected (Cmax 492.7 ± 35.5 ng mL-1), followed by C6G (Cmax 96.1 ± 33.8 ng mL-1) and M6G (Cmax 22.3 ± 4.96 ng mL-1). Morphine and norcodeine were the least abundant metabolites with Cmax of 3.17 ± 0.95 and 1.42 ± 0.79 ng mL-1, respectively. No significant adverse or excitatory effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Following oral administration, codeine is rapidly metabolized to morphine, M3G, M6G, C6G and norcodeine in horses. Plasma concentrations of M6G, a presumed active metabolite of morphine, were comparable to concentrations reported previously following administration of an analgesic dose of morphine to horses. Codeine was well tolerated based on pharmacodynamic variables and behavioral observations.

Detection of codeine and fentanyl in saliva, blood plasma and whole blood in 5-minutes using a SERS flow-separation strip.

A simple-to-use device to measure drugs in saliva, blood plasma, and whole blood for point-of-care analysis and treatment of overdose patients has been investigated. A rudimentary flow strip has been developed to separate opioids from these biofluids for analysis by surface-enhanced Raman spectroscopy (SERS). The strips are based on lateral flow assays, in which the antibodies have been substituted by SERS-active pads for detection. Samples of codeine and fentanyl, artificially added to these biofluids, were measured using the strips by a field-usable Raman spectrometer. We report measurement of these drugs in these biofluids from 0.5 to 5 µg mL-1 in 5 minutes. Calculated limits of detection for the spectra suggest that these drugs could be measured at 5 to 20 ng mL-1 with improvements in the strips' separation capability.

Quantitative detection of codeine in human plasma using surface-enhanced Raman scattering via adaptation of the isotopic labelling principle.

In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q2). In addition, the employment of codeine-d6 as the internal standard further improved the accuracy of the model, by increasing the Q2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm-1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 µM (209.55 ng mL-1) and 1.39 µM (416.12 ng mL-1), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.

Determination of pholcodine in syrups and human plasma using the chemiluminescence system of tris(1,10 phenanthroline)ruthenium(II) and acidic Ce(IV).

Pholcodine is an opiate derivative drug which is widely used in pediatric medicine. In this study, a chemiluminescence (CL) method is described that determines pholcodine in human plasma and syrup samples. This method is based on the fact that pholcodine can greatly enhance the weak CL emission of reaction between tris(1,10 phenanthroline)ruthenium(II), Ru(phen)32+ , and acidic Ce(IV). The CL mechanism is described in detail using UV-vis light, fluorescence and CL spectra. Effects of chemical variables were investigated and under optimum conditions, CL intensity was proportional to the pholcodine concentration over the range 4.0 × 10-8 to 8.0 × 10-6  mol  L-1 . The limit of detection (LOD) (S/N = 3) was 2.5 × 10-8  mol  L-1 . Percent of relative standard deviations (%RSD) for 3.0 × 10-7 and 3.0 × 10-6  mol  L-1 of pholcodine was 2.9 and 4.0%, respectively. Effects of common ingredients were investigated and the method was applied successfully to the determination of pholcodine in syrup samples and human plasma.

Physiologically based pharmacokinetic modeling revealed minimal codeine intestinal metabolism in first-pass removal in rats.

The physiologically based model with segregated flow to the intestine (SFM-PBPK; partial, lower flow to enterocyte region vs. greater flow to serosal region) was found to describe the first-pass glucuronidation of morphine (M) to morphine-3ß-glucuronide (MG) in rats after intraduodenal (i.d.) and intravenous (i.v.) administration better than the traditional model (TM), for which a single intestinal flow perfused the whole of the intestinal tissue. The segregated flow model (SFM) described a disproportionately greater extent of intestinal morphine glucuronidation for i.d. vs. i.v. administration. The present study applied the same PBPK modeling approaches to examine the contributions of the intestine and liver on the first-pass metabolism of the precursor, codeine (C, 3-methylmorphine) in the rat. Unexpectedly, the profiles of codeine, morphine and morphine-3ß-glucuronide in whole blood, bile and urine, assayed by LCMS, were equally well described by both the TM-PBPK and SFM-PBPK. The fitted parameters for the models were similar, and the net formation intrinsic clearance of morphine (from codeine) for the liver was much higher, being 9- to 13-fold that of the intestine. Simulations, based on the absence of intestinal formation of morphine, correlated well with observations. The lack of discrimination of SFM and TM with the codeine data did not invalidate the SFM-PBPK model but rather suggests that the liver is the only major organ for codeine metabolism. Because of little or no contribution by the intestine to the metabolism of codeine, both the TM- and SFM-PBPK models are equally consistent with the data. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetics and pharmacodynamics of oral acetaminophen in combination with codeine in healthy Greyhound dogs.

The purpose of this study was to determine the pharmacokinetic and antinociceptive effects of an acetaminophen/codeine combination administered orally to six healthy greyhounds. Antinociception was assessed using an electronic von Frey (vF) device as a mechanical/pressure model. Acetaminophen was administered at a dose of 600 mg (14.4-23.1 mg/kg) and codeine phosphate at 90 mg (2.1-3.3 mg/kg) equivalent to 67.5 mg codeine base (1.6-2.5 mg/kg). The geometric mean maximum plasma concentrations of acetaminophen, codeine, and codeine-6-glucuronide were 7.95 µg/mL, 11.0 ng/mL, and 3819 ng/mL, respectively. Morphine concentrations were <1 ng/mL. The terminal half-lives of acetaminophen, codeine, and codeine-6-glucuronide were 0.94, 1.71, and 3.12 h. There were no significant changes in vF thresholds, except at 12 h which decreased on average by 17% compared to baseline. The decrease in vF thresholds at 12 h could be due to aversion, hyperalgesia, or random variability. The lack of antinociception in this study could be due to a true lack of antinociception, lack of model sensitivity, or specificity. Further studies using different models (including clinical trials), different dog breeds, multiple dose regimens, and a range of dosages are needed prior to recommended use or concluding lack of efficacy for oral acetaminophen/codeine in dogs.

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.

The impact of CYP2D6 polymorphisms on the pharmacokinetics of codeine and its metabolites in Mongolian Chinese subjects.

PURPOSE: Codeine is an analgesic drug acting on µ-opioid receptors predominantly via its metabolite morphine formed almost exclusively by CYP2D6. Genetic polymorphisms in CYP2D6 are associated with diminished pain relief and/or severe opioid side effects. In Chinese individuals, CYP2D6*10 is the most common allele with reduced enzyme activity. In this study, we investigated the effect of this allele on the pharmacokinetics of codeine and its metabolites. METHOD: A blood sample was collected from healthy Mongolian volunteers for CYP2D6 genotyping using a PCR-RFLP assay. A pharmacokinetic study was then carried out in three groups with CYP2D6*1/*1 (n=10), CYP2D6*1/*10 (n=10) and CYP2D6*10/*10 (n=9) genotypes by collecting serial blood samples for determination of plasma levels of codeine and its metabolites, morphine, morphine 3-glucuronide (M3G) and morphine 6-glucuronide (M6G) before and after a single 30-mg oral dose of codeine phosphate. Codeine and its metabolites were measured by LC-MS/MS. RESULTS: No significant differences were observed in the pharmacokinetic parameters of codeine in the three genotype groups. However, the C( max) and AUC(0-∞) of morphine, M3G and M6G were significantly different between the study groups (P<0.05). Compared with the *1/*1 group, the AUC(0-∞) for morphine in the *1/*10 and *10/*10 groups decreased by ratios (95 % CI) of 0.93 (0.26-1.59) and 0.494 (0.135-0.853) respectively. Corresponding ratios for M3G were 0.791 (0.294-1.288) and 0.615 (0.412-0.818) and for M6G were 0.643 (0.39-0.957) and 0.423 (0.267-0.579). CONCLUSION: This study demonstrates that the CYP2D6*10 allele plays an important role in the pharmacokinetics of the O-demethylated metabolites of codeine after oral administration.

A near-infrared plasmonic biosensor for detection of morphine and codeine in biological samples based on the end-to-end assembly of modified gold nanorods.

The analytical determination of opiates in biological samples is a critical mission and remains a challenge for almost all judicial and clinical drug testing panels due to their high abuse potential. Based on the high sensitivity of the longitudinal surface plasmon resonance (LSPR) peak of gold nanorods (AuNRs), we successfully developed a novel and simple refractive index sensing platform for detection of morphine (MOR) and codeine (COD) by means of 2-amino-5-mercapto-1,3,4-thiadiazole functionalized gold nanorods (AMTD-AuNRs) in aqueous solution, which is, to the best of our knowledge, the first report on the assay of MOR and COD using AuNRs. AMTD molecules strongly anchor onto the tips of AuNRs via the mercapto group and subsequent hydrogen-bonding interactions between AMTD and the analytes induced end-to-end chain assembly of AuNRs and a consequent decrease of the LSPR absorption band at 850 nm along with a bathochromic shift and emergence of a new hybridized plasmon mode at 1050 nm which was characterized using a Vis-NIR spectrophotometer. After systematic optimization, the absorbance ratio (A1050/A850) was proportional to the concentration of MOR in the ranges of 0.08-5 µM and 0.2-8 µM for COD without any significant effect from possible interferents. Furthermore, detection limits of 40 and 62 nM were achieved for MOR and COD, respectively, which are much lower than the cut-off level of 2000 ng mL-1 for opiates in urine samples set by the Substance and Abuse Mental Health Services Administration (SAMHSA). Eventually, as proof-of-applicability, human urine and blood serum samples spiked with MOR and COD were analyzed and excellent recoveries ranging from 94.4 to 108.9% were obtained, demonstrating the successful applicability of the designed refractive index probe in real biological specimens.

Toxicogenetics--cytochrome P450 microarray analysis in forensic cases focusing on morphine/codeine and diazepam.

Genetic polymorphisms in cytochrome P 450 (CYP) enzymes could lead to a phenotype with altered enzyme activity. In pharmacotherapy, genotype-based dose recommendations achieved great importance for several drugs. In our pilot study, we ask if these genetic tests should be applied to forensic problems as a matter of routine. Starting from 2004 through 2008, we screened routine cases for samples where the relation of parent compound to metabolite(s) (P/M ratio), particularly morphine to codeine ratios and diazepam to its metabolites, was noticeable or not consistent with the information provided by the defendants. We found 11 samples with conspicuous results. These were analyzed for polymorphisms of the CYP 2D6 and 2C19 genes using the Roche AmpliChip Cytochrome P450 Genotyping test. If not previously conducted, a general unknown analysis by gas chromatography/mass spectrometry (GC/MS) was additionally carried out. For CYP 2D6, we found two cases with the genotype poor metabolizer (PM), three cases with heterozygote extensive metabolizer genotype classified as an intermediate metabolizer (IM) with probably reduced enzyme activities, but no ultrarapid metabolizer genotype. For CYP 2C19, two cases were characterized as IM phenotypes, with no PM found. Once we achieved no appropriate amounts of DNA, one case was excluded after GC/MS analysis. Only in one case could the polymorphism clearly explain the changes in drug metabolism. More frequently, a drug-drug interaction was thought to have a stronger impact. Additionally, our results suggest that IM genotypes may be more relevant than previously suspected. With respect to the small number of cases in which we thought a genotyping would be helpful, we conclude that the overall relevance of toxicogenetics in forensic problems is moderate. However, in some individual cases, a genotyping may provide new insight.

Is there a role for therapeutic drug monitoring with codeine?

Codeine is an old and commonly used analgesic agent for mild to moderate pain. It is the prototypical "prodrug" in that its analgesic effect is almost wholly dependent on its biotransformation to morphine, a process that is mediated by the polymorphic cytochrome P450 2D6 enzyme. As such, interindividual variability in codeine metabolism and response is a clinical reality, and there has been much progress in characterizing the genetic causes of this variability in diverse populations. Yet despite the potential for both life-threatening adverse reactions and lack of therapeutic effect, codeine is not commonly indicated for therapeutic drug monitoring. This review will discuss the relative role of pharmacogenetics and therapeutic drug monitoring in predicting and/or maintaining adequate and safe analgesia with codeine. The review will end on a discussion of how the marriage of these 2 fields may provide new insights into the mechanisms of codeine-induced toxicity and analgesia.

Determination of morphine, codeine, and paclitaxel in human serum and plasma by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography method is proposed for the determination of morphine, codeine, and paclitaxel at clinical relevant levels in human serum and plasma, which are employed in the treatment of patients with cancer. Optimal conditions for the separation were investigated. A background electrolyte solutions consisting of 20 mM borate buffer adjusted to pH 8.5, sodium dodecyl sulphate 60 mM and 15% methanol, hydrodynamic injection, and 25 kV as separation voltage were used. Detection wavelength was 212 nm for morphine and codeine and 200 nm for paclitaxel. Aspects such as stability of the solutions, linearity, accuracy, precision, and robust and ruggedness were examined in order to validate the proposed method. Detection limits obtained for all the studied compounds ranged between 26 and 52 ng/mL. Before micellar electrokinetic chromatography determination, the samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C(18) cartridge. This method was applied to the analysis of serum and plasma samples from different cancer patients undergoing treatment with paclitaxel or/and codeine.

Postmortem Brain-Blood Ratios of Codeine, Fentanyl, Oxycodone and Tramadol.

The analgesics, codeine, fentanyl, oxycodone and tramadol, frequently occur in postmortem cases and determining their role in the cause of death can be challenging. However, postmortem blood is susceptible to redistribution and may not be available in cases of severe blood loss, putrefaction or burns. Brain tissue may serve as a viable supplement to blood or on its own, as it is resistant to postmortem redistribution and often available as a sample matrix when blood is not available. We present brain and blood concentrations and brain-blood ratios of the four analgesics from 210 autopsy cases. The cases were classified according to the presumed cause of death: A: The compound was believed to have solely caused a fatal intoxication. B: The compound was assumed to have contributed to a fatal outcome in combination with other drugs, alcohol or disease. C: The compound was not regarded as being related to the cause of death. Blood and brain samples were prepared by automatic solid phase extraction and quantified by liquid chromatography-mass spectrometry. The squared correlation coefficients between concentrations in brain tissue and blood ranged 0.45-0.91. The median brain-blood ratios were codeine 1.8 (range 0.47-4.6), fentanyl 2.1 (range 0.29-16), oxycodone 1.8 (range 0.11-6.0) and tramadol 1.8 (range 0.047-6.8). A significantly higher brain-blood ratio of codeine was observed in cases where heroin had been administered, although there was a wide overlap. Intravenous and transdermal fentanyl administration could not be distinguished based on the blood or brain concentration or the brain-blood ratio. The results of this study may benefit the toxicological investigation in postmortem cases where one of the four analgesics are suspected of having contributed to or caused a fatal intoxication.

Toxicological detection of pholcodine in blood, urine and hair in three cases of fatal intoxication.

Pholcodine is an opioid antitussive reputed for its low toxicity and absence of addictive effect. We report three cases of pholcodine intoxication with fatal outcome. Large concentrations of pholcodine were quantified by gas chromatography coupled to mass spectrometry (GC/MS) in peripheral postmortem blood (respectively 2890 ng/mL, 979 ng/mL and 12,280 ng/mL). Segmental hair analyses by GC/MS and detected pholcodine in three 1.5-2 cm segments (38-161 ng/mg, 8.54-41.6 ng/mg, and 0.26-2.66 ng/mg, respectively). These findings underline that pholcodine can be involved in fatal poisoning and raise the question of misuse or abuse and of taking account of this drug in opioid overdose prevention policies.

Forensic toxicology findings in deaths involving gamma-hydroxybutyrate.

Concentrations of the illicit drug gamma-hydroxybutyrate (GHB) were determined in femoral venous blood and urine obtained at autopsy in a series of GHB-related deaths (N = 49). The analysis of GHB was done by gas chromatography after conversion to gamma-butyrolactone and quantitation of the latter with a flame ionization detector. The cutoff concentration of GHB in femoral blood or urine for reporting positive results was 30 mg/L. The deceased were mainly young men (86%) aged 26.5 +/- 7.2 years (mean +/- SD), and the women (14%) were about 5 years younger at 21.4 +/- 5.0 years. The mean, median, and highest concentrations of GHB in femoral blood (N = 37) were 294, 190, and 2,200 mg/L, respectively. The mean urine-to-blood ratio of GHB was 8.8, and the median was 5.2 (N = 28). In 12 cases, the concentrations of GHB in blood were negative (<30 mg/L) when the urine contained 350 mg/L on average (range 31-1,100 mg/L). Considerable poly-drug use was evident in these GHB-related deaths: ethanol (18 cases), amphetamine (12 cases), and various prescription medications (benzodizepines, opiates, and antidepressants) in other cases. Interpreting the concentrations of GHB in postmortem blood is complicated because of concomitant use of other psychoactive substances, variable degree of tolerance to centrally acting drugs, and the lack of reliable information about survival time after use of the drug.

Influence of the CYP2D6 polymorphism and hemodialysis on codeine disposition in patients with end-stage renal disease.

OBJECTIVE: We studied the influence of three factors on drug disposition: genetic polymorphism, impaired renal excretion of drug metabolites, and the possible elimination by hemodialysis (HD), using codeine as a model drug. METHODS: Based on the genotyping of three CYP2D6 polymorphisms in 228 HD patients, nine extensive metabolizers (EMs) and two poor metabolizers (PMs) were given a single oral dose of 50 mg codeine phosphate. Plasma concentrations of its metabolites codeine-6-glucuronide (C6G), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were determined after 2, 4, 6, 8 and 24 h (beginning of the HD session) and again after 4 h of HD (28 h). Codeine metabolites in plasma were quantitated by liquid chromatography-mass spectrometry (LC-MS). RESULTS: The concentrations of C6G in plasma were high and similar in EMs and PMs. Two hours after the codeine intake, the mean concentration of M3G was 210 nM in EMs vs. 3.5 nM in PMs. The M6G metabolite concentrations could be quantitated in EMs but were below the limit of quantification in PMs (<1 nM). All three codeine metabolites/glucuronides remained unchanged or even increased until the start of HD, and thereafter, the concentrations decreased dramatically during the HD procedure. CONCLUSIONS: Formation of the codeine metabolites M3G and M6G was dependent on the CYP2D6 genotype, as previously shown in healthy individuals. Elimination of glucuronides in these patients was absent until HD was performed. These factors need to be taken into consideration when drugs metabolized by CYPs are prescribed in HD patients.

Rapid simultaneous determination of codeine and morphine in plasma using LC-ESI-MS/MS: application to a clinical pharmacokinetic study.

A rapid and sensitive high-performance LC-MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid-liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C(18 )column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H](+ )ions, mass-to-charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2-100/0.5-250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC-MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.

Quantitative analysis of desomorphine in blood and urine using solid phase extraction and gas chromatography-mass spectrometry.

Desomorphine, a semi-synthetic opioid, is a component of the street drug Krokodil. Despite continued reports of Krokodil use, confirmation via toxicological testing remains scarce. The lack of confirmed desomorphine reports may be in part due to the limited published analytical methodology capable of detecting desomorphine at forensically relevant concentrations. In an effort to assist with identification efforts, a robust analytical method was developed and validated. Solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) were used to determine desomorphine in blood and urine using a deuterated analog as the internal standard. Data was acquired using selected ion monitoring (SIM) mode. Extraction efficiencies in blood and urine were 69% and 90%, respectively. The limits of quantitation in blood and urine were 5 ng/mL and 8 ng/mL, ten-fold lower than previously published methods. Intra- and inter-assay CVs were 2-4% (n = 3) and 3-7% (n = 15), respectively. The method was fully validated in accordance with published guidelines for forensic use. Furthermore, it provides a means by which desomorphine can be identified in toxicology specimens at forensically relevant concentrations, without the need for derivatization.

Stability studies in biological fluids during post-analysis custody. Opiate compounds derived from heroin consumption.

In Forensic Toxicology, the evidences have to be maintained under custody for, at least, one year. Depending on the conditions and duration of storage, drug concentrations might have changed considerably since the first analysis. The aim of this study is to evaluate in vitro stability of opiate compounds, derived from heroin consumption, 6-acetylmorphine (6-MAM), morphine (MOR) and codeine (COD), in blood and urine, during post-analysis custody. Parameters evaluated were: time of custody, temperature, addition of preservative (blood) and pH (urine). Blood and urine samples were spiked with the three analytes to give a final concentration of 1000 ng/mL. The prepared samples were divided into 2 groups and stored at two temperatures (4 °C and -20 °C). Each one of these groups was subsequently divided in other two groups: with and without preservative (1%NaF) for blood, and pH 4 and 8 in the case of urine. 6-MAM, MOR and COD were analyzed by GCMS after SPE and derivatization with BSTFA. Analyses were performed in triplicate every two weeks for a year. In blood samples 6-MAM is the only compound that degrades. The best storage conditions were at -20 °C with NaF, with 6-MAM recoveries, after one year of custody, of 47.1 ± 1.5%; while in the other conditions 6-MAM disappeared after 215 days (at 4 °C with NaF), 45 days (at -20 °C without NaF) and 15 days (at 4 °C without preservative). COD does not degrade, with recoveries higher than 90%, in all of the conditions. They ranged from 89.7 ± 3.6% in samples maintained at -20 °C without NaF to 95.9 ± 2.0% in those maintained at 4 °C with NaF. MOR recoveries were lower than those of COD. They ranged from 66.9 ± 3.6%, in frozen samples added with NaF, to 78.6 ± 0.5% in refrigerated samples without preservative. In urine samples the three compounds were stable in all the studied conditions, with the exception of 6-MAM in samples at pH 8 and stored at 4 °C. In these conditions, 6-MAM disappeared after 135 days of custody; while recoveries in the other conditions ranged from 93.7 ± 6.4%, at 4 °C and pH 4, to 85.1 ± 2.0% at -20 °C and pH 8. MOR and COD recoveries were similar in the four conditions. In the case of MOR, they ranged from 82.1 ± 1.2% at 4 °C and pH 4 to 89.5 ± 6.0% at -20 °C and pH 8. As far as COD is concerned, recoveries ranged from 111.6 ± 5.8% at 4 °C and pH 8 to 102.6 ± 1.2% at 4 °C and pH 4. In conclusion, the study showed that the most labile opiate compound is 6-MAM. Its stability mainly depends on urine pH or the addition of preservative, in blood samples. The best storage conditions for samples from heroin consumers are in the freezer, at -20 °C. In addition, blood samples must be added with 1%NaF and urine samples must be buffered at pH 4.