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.

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.

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.

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.

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.

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.

Ultra-sensitive electrochemical sensing of acetaminophen and codeine in biological fluids using CuO/CuFe2O4 nanoparticles as a novel electrocatalyst.

Copper ferrite-copper oxide (CuO-CuFe2O4) nanoparticles as a semiconductor composite with p-n junction were synthesized by co-precipitation reaction. Then, a novel CuO-CuFe2O4 carbon paste modified electrode was fabricated which displays an effectual electrocatalytic response to the oxidation of acetaminophen (AC) and codeine (CO). A linear range of 0.01-1.5 µmol L-1 and 0.06-10.0 µmol L-1 with the detection limits of 0.007 µmol L-1 and 0.01 µmol L-1 were achieved for AC and CO, respectively. The practical usage of the proposed sensor revealed reasonable results for quantification of AC and CO in biological fluids.

Metabolites of Heroin in Several Different Post-mortem Matrices.

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone.

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an Athlete Biological Passport (ABP). The aim of this study was to investigate the possible influence of short-term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co-administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On Day 3, 500 mg or 125 mg of testosterone enanthate (Testoviron®-Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6, and 14 days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20% after 3 days' use of codeine (p = 0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75% of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion, our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.

3D spongy graphene-modified screen-printed sensors for the voltammetric determination of the narcotic drug codeine.

Adenine-functionalized spongy graphene (FSG) composite, fabricated via a facile and green synthetic method, has been explored as a potential electrocatalyst toward the electroanalytical sensing of codeine phosphate (COD). The synthesized composite is characterized using Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray powder diffraction, UV-vis absorption spectroscopy, scanning electron microscopy, high resolution transmission electron microscopy (HRTEM), and thermogravimetric analysis. The FSG was electrically wired via modification upon screen-printed (macro electrode) sensors, which behave as a hybrid electrode material for the sensitive and selective codeine phosphate (COD) determination in the presence of paracetamol (PAR) and caffeine (CAF). The FSG- modified sensor showed an excellent electrocatalytic response towards the sensing of COD with a wide linear response range of 2.0 × 10-8-2.0 × 10-4M and a detection limit (LOD) of 5.8 × 10-9M, indicating its potential for the sensing of COD in clinical samples and pharmaceutical formulations.

Fast quantitation of opioid isomers in human plasma by differential mobility spectrometry/mass spectrometry via SPME/open-port probe sampling interface.

Mass spectrometry (MS) based quantitative approaches typically require a thorough sample clean-up and a decent chromatographic step in order to achieve needed figures of merit. However, in most cases, such processes are not optimal for urgent assessments and high-throughput determinations. The direct coupling of solid phase microextraction (SPME) to MS has shown great potential to shorten the total sample analysis time of complex matrices, as well as to diminish potential matrix effects and instrument contamination. In this study, we demonstrate the use of the open-port probe (OPP) as a direct and robust sampling interface to couple biocompatible-SPME (Bio-SPME) fibres to MS for the rapid quantitation of opioid isomers (i.e. codeine and hydrocodone) in human plasma. In place of chromatography, a differential mobility spectrometry (DMS) device was implemented to provide the essential selectivity required to quantify these constitutional isomers. Taking advantage of the simplified sample preparation process based on Bio-SPME and the fast separation with DMS-MS coupling via OPP, a high-throughput assay (10-15 s per sample) with limits of detection in the sub-ng/mL range was developed. Succinctly, we demonstrated that by tuning adequate ion mobility separation conditions, SPME-OPP-MS can be employed to quantify non-resolved compounds or those otherwise hindered by co-extracted isobaric interferences without further need of coupling to other separation platforms.

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.

Simultaneous determination of ascorbic acid, acetaminophen and codeine based on multi-walled carbon nanotubes modified with magnetic nanoparticles paste electrode.

Based on incorporating ZnCrFeO4 into multi-walled carbon nanotubes paste matrix (MWCNTs/ZnCrFeO4/CPE), a chemically modified electrode was prepared for the simultaneous determination of ascorbic acid (AA), acetaminophen (AC) and codeine (CO). The prepared electrode, MWCNTs/ZnCrFeO4/CPE, was characterized by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The MWCNTs/ZnCrFeO4/CPE showed an efficient electrocatalytic activity for the oxidation of AA, AC, and CO. The separations of the oxidation peak potentials for AA-AC and AC-CO were about 250mV and 630mV, respectively. The calibration curves obtained for AA, AC, and CO were in the ranges of 0.4-730.0µmolL(-1), 0.1-368.0µmolL(-1), and 0.3-250.0µmolL(-1), respectively. The detection limits (S/N=3) were 0.03µmolL(-1), 0.009µmolL(-1), and 0.01µmolL(-1) for AA, AC, and CO, respectively. The method was also successfully employed as a selective, simple, and precise method to determinate AA, AC, and CO in pharmaceutical and biological samples.

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.

Influence of Sampling Procedure on Codeine Concentrations in Oral Fluid.

For many drugs, there is a poor correlation between the plasma and oral fluid (OF) concentrations, due to differences in OF pH, oral contamination, stimulation of OF flow and variability of the volume of sample taken. The aim of this study was to evaluate the OF/plasma ratio and variability in drug concentration in OF sampled by two commercially available collection systems: Saliva Collection System (SCS) and Quantisal. Blood and OF samples were collected from 12 volunteers after intake of 19.5 mg codeine phosphate. Six persons were sampled by SCS first, followed by Quantisal; six other participants used Quantisal before SCS. The OF content of SCS tubes was measured spectrophotometrically. The Quantisal devices were weighed to correct for the effectively obtained OF volume. Codeine was measured by gas chromatography-mass spectrometry. The mean codeine concentration at 1 h was 29.8 ± 18.8 µg/L in plasma, 72.8 ± 63.9 µg/L in SCS OF and 85.3 ± 72.6 µg/L in Quantisal OF. The mean OF/plasma ratio was 2.30 ± 0.77 (SCS) and 2.69 ± 1.94 (Quantisal). Pearson's correlation coefficient between OF and plasma codeine concentrations was statistically significantly (P = 0.005) higher for SCS (R(2) = 0.745) than for Quantisal (R(2) = 0.403). The variability in ratios with Quantisal was markedly reduced when used after SCS. Codeine concentrations measured in OF taken with SCS correlate better with plasma concentrations than in OF obtained with Quantisal, particularly when Quantisal was used first.

UPLC-MS-MS Determination of Dihydrocodeine in Human Plasma and Its Application to a Pharmacokinetic Study.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method was developed to determine dihydrocodeine (DHC) in human plasma using diazepam as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by multiple reaction monitoring (MRM) of the transitions at m/z 302.3 → 199.2 for DHC and m/z 285.1 → 193.1 for IS. The linearity of this method was found to be within the concentration range of 0.5-100 ng/mL with a lower limit of quantification of 0.5 ng/mL. The overall run time was 4.0 min. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of DHC in healthy Chinese volunteers after oral administration.

A new DBS card with spot sizes independent of the hematocrit value of blood.

BACKGROUND: DBS cards have been a big promise for decades. However, blood with low hematocrit (Ht) values results on regular cellulose-based DBS cards in larger spot sizes, compared with blood with high Ht-values. A new material has been developed to solve this problem. RESULTS: This material, based on hydrophilic-coated woven polyester fibers, shows spot sizes independent of the Ht-value of blood. Homogeneity over the spot is within 10% RSD. CONCLUSION: Quantitative measurements over a broad Ht range show nonbiased results compared with whole spot analysis. The cards are experienced as reproducible, robust and easy to use on aspects of punchability and extractability.