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High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.

Chromatography, High Pressure Liquid↗

A double-blind randomised controlled clinical trial of the effect of preoperative ibuprofen, diclofenac, paracetamol with codeine and placebo tablets for relief of postoperative pain after removal of impacted third molars.

We conducted a randomised double-blind placebo-controlled single-centre study to compare the effect of preoperative ibuprofen 600 mg, diclofenac 100 mg, paracetamol 1 g with codeine 60 mg or placebo (Vitamin C 50 mg) tablets for relief of postoperative pain in 119 patients who had day case operations under general anaesthesia for removal of impacted third molars. Patients were given the tablets 1 h before operation. Pain was assessed using visual analogue scales and verbal rating scales preoperatively at 15 and 30 min and 1 and 3 h postoperatively. After they had gone home, patients were contacted by telephone at 6 and 24 h postoperatively to find out whether they had any adverse effects from the analgesics. There was no significant difference in the extent of postoperative pain among the four groups, but the placebo group had significantly shorter times before their first request for postoperative analgesics (median 17 min, range 14-90) than the diclofenac group (median 32, range 15-150). Preoperative analgesics at the stated doses are effective in providing immediate postoperative pain control after operations on third molars. There were, however, some side-effects including nausea, vomiting, headaches, and gastrointestinal discomfort, but there were no significant differences among the active analgesic groups with respect to adverse events either shortly after operation or at 6 or 24 h.

Acetaminophen↗

Opiate receptor binding properties of morphine-, dihydromorphine-, and codeine 6-O-sulfate ester congeners.

A series of 3-O-acyl-6-O-sulfate esters of morphine, dihydromorphine, N-methylmorphinium iodide, codeine, and dihydrocodeine were prepared and evaluated for their ability to bind to mu-, delta-, kappa(1)-, kappa(2)-, and kappa(3)-opiate receptors. Several compounds exhibited good affinity for the mu-opiate receptor. Morphine-3-O-propionyl-6-O-sulfate had four times greater affinity than morphine at the mu-opiate receptor and was the most selective compound at this receptor subtype.

Animals↗

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography.

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.

Cations↗

Development and validation of a capillary electrophoresis method for the determination of codeine, diphenhydramine, ephedrine and noscapine in pharmaceuticals.

The present work describes a simple, accurate and rapid method for the separation and simultaneous determination of codeine, diphenhydramine, ephedrine and noscapine present in cough-cold syrup formulations by capillary zone electrophoresis. Factors affecting the separation were the buffer pH and concentration, applied voltage, and presence of additives. Separations were carried out in less than 10 min with a 20 mM sodium tetraborate buffer, pH 8.50. The carrier electrolyte gave baseline separation with good resolution, great reproducibility and accuracy. Calibration plots were linear over at least three orders of magnitude of analyte concentrations, the lower limits of detection being within the range 0.42-1.33 microg ml(-1). Detection was performed by UV absorbance at wavelengths of 205 and 250 nm. Quantification of the components in actual syrup formulations was calculated against the responses of freshly prepared external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control. The procedure was fast and reliable and commercial pharmaceuticals could be analyzed without prior sample clean-up procedure.

Antitussive Agents↗

Evidence for CYP2D1-mediated primary and secondary O-dealkylation of ethylmorphine and codeine in rat liver microsomes.

The purpose of the present study was to investigate the role of specific CYPs responsible for the O-dealkylation of ethylmorphine (EM) and codeine (CD) to morphine (M), as well as that of norethylmorphine (NEM) and norcodeine (NCD) to normorphine (NM) in rat liver microsomes. Liver microsomes metabolize EM and CD to M, and NEM and NCD to NM, in the presence of an NADPH-generating system. The metabolites of EM and CD were determined by HPLC with UV and electrochemical detection. In the present study, the role of CYP2D1 in O-dealkylation of EM/NEM and CD/NCD was investigated by use of specific antiCYP antibodies. When testing rabbit antirat CYP2D1, 2E1, 2C11, and 3A2 antibodies, only the antiCYP2D1 antibody inhibited the EM/NEM and CD/NCD O-dealkylase activities significantly. The maximum inhibition achieved was approximately 80% at a protein ratio (IgG to microsomes) of 10:1, p = 0.001. The contribution of CYP2D1 to the O-dealkylation of EM/NEM and CD/NCD was further confirmed by use of the specific CYP2D1 inhibitors quinine and propafenone. Five microM of quinine inhibited the EM/NEM and CD/NCD O-dealkylase activities by approximately 80%. The CYP3A inhibitor troleandomycin (TAO) failed to inhibit the CYP2D1 catalyzed reaction, but did inhibit the N-demethylation of EM and CD. The O-dealkylation of NEM and NCD was also impaired in Dark Agouti rat (DA) liver microsomes. Taken together, the immunoinhibition and chemical-inhibitor studies of rat liver microsomes provided convincing evidence for the involvement of CYP2D1, the rat counterpart of human CYP2D6, in the metabolism of EM/NEM and CD/NCD to the corresponding O-dealkylated metabolites.

Animals↗

Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate.

Morphine-6-sulfate (M6S) and codeine-6-sulfate (C6S) are mu-selective opiates which have been isolated from brain. M6S is an effective analgesic, with a 30-fold greater potency than morphine in the mouse radiant heat tailflick assay and similar to the active morphine metabolite morphine-6beta-glucuronide (M6G). M6S analgesia is reversed by 3-methoxynaltrexone at low antagonist doses which are inactive against morphine, suggesting that M6S may be acting through the same mechanisms as M6G. Consistent with this possibility, antisense mapping of the MOR-1 clone revealed that M6S analgesia was lowered by probes targeting exon 2 and not by targeting exon 1, a sensitivity profile similar to that of M6G and not morphine. C6S also has analgesic activity at doses approximately 10-fold greater than M6S. However, its characterization was impeded by the appearance of seizures at doses below full analgesic activity. Thus, M6S is a potent analgesic with pharmacological properties similar to M6G. C6S has limited utility due to its high level of toxicity.

Analgesics, Opioid↗

Separation of ibuprofen, codeine phosphate, their degradation products and impurities by capillary electrophoresis. I. Method development and optimization with fractional factorial design.

A capillary electrophoresis method has been developed and optimized for the separation of ibuprofen, codeine phosphate and their main degradation products and impurities. In the course of developing the method, it was found that micellar electrokinetic capillary chromatography was necessary for the separation of the eleven peaks. A fractional factorial design was used for the optimization of the experiments. Six process parameters were varied at two levels: the concentration of sodium dodecyl sulfate (SDS), the pH, the concentration of acetonitrile, the concentration of boric acid, the field strength and the temperature. All these factors had a significant effect on the migration time and resolution. The optimum conditions were found to be a borate buffer of 40 mM H3BO3 at pH 10 with the addition of 40 mM SDS and 9% acetonitrile, a field strength of 515 V/cm and a temperature of 25 degrees C. This resulted in baseline separation of the eleven peaks within 12 min.

Acetonitriles↗

Separation of ibuprofen, codeine phosphate, their degradation products and impurities by capillary electrophoresis. II. Validation.

A micellar electrokinetic chromatography method for the determination of ibuprofen and codeine phosphate hemihydrate and their degradation products and impurities in a commercial tablet formulation has been validated. The validation has been performed according to the International Conference of Harmonisation's guidance on the validation of analytical methods, and selectivity, linearity, accuracy, precision, detection limit, quantitation limit, robustness and range test were performed to determine the suitability of the method. It was possible to use the fractional factorial design model from the optimisation of the method to draw conclusions about its robustness. The results confirm that the method is highly suitable for its intended purpose.

Chemistry, Pharmaceutical↗

Full and fractionated experimental designs for robustness testing in the high-performance liquid chromatographic analysis of codeine phosphate, pseudoephedrine hydrochloride and chlorpheniramine maleate in a pharmaceutical preparation.

This paper describes the testing of a saturated factorial design using a full factorial design. Saturated factorial designs are often used to test the robustness of high-performance liquid chromatography (HPLC) methods, however they are based on several assumptions. A full factorial design relies on fewer assumptions and hence could be used to evaluate the effectiveness of the saturated design. Both designs were used to test a gradient HPLC method for the assay of codeine phosphate, pseudoephedrine hydrochloride and chlorpheniramine maleate. Six HPLC conditions, including wavelength, mobile phase pH and ion pairing reagent concentration were tested using the saturated design. Three of these factors were selected for full evaluation using a full factorial design. The results showed that the main effects calculated by each design were comparable. However, the saturated design showed higher standard errors, probably due to the effects of changing several more factors. One interaction effect was indicated as a confounding effect by the saturated design and this was confirmed by the calculation of the same interaction effect using the full design. Overall the method was shown to be robust under the variety of HPLC conditions tested.

Chlorpheniramine↗

Endogenous codeine and morphine in anorexia and bulimia nervosa.

The endogenous plasma alkaloids codeine and morphine were shown to be elevated in patients with anorexia nervosa and bulimia nervosa compared to control subjects. The role of these opioids in the pathophysiology of these eating disorders is discussed in relation to an auto-addiction opioid model. This model proposes that endogenous opioids are released during an initial period of dieting and reinforce a state of starvation dependence [1,2].

Adolescent↗

Codeine intoxication in childhood.

Clinical reports on 430 children with acute codeine intoxication are evaluated. Of 234 children who had taken more than 5 mg/kg body-weight, 8 had respiratroy arrest necessitating intubation and artificial ventilation; 2 of them died. In all other cases the intoxication produced one or more of the following symptoms: somnolence, ataxia, miosis, vomiting, rash, swelling, and itching of the skin, but no life-threatening side-effects. Close supervision of respiration is the main principle of management when more than 2 mg codeine/kg body-weight has been taken. Gastric voiding may be useful if done soon after ingestion. Charcoal and purgatives should be given in all cases.

Antitussive Agents↗

Single-patient data meta-analysis of 3453 postoperative patients: oral tramadol versus placebo, codeine and combination analgesics.

The analgesic effectiveness and safety of oral tramadol were compared with standard analgesics using a meta-analysis of individual patient data from randomised controlled trials in patients with moderate or severe pain after surgery or dental extraction. Calculation of %maxTOTPAR from individual patient data, and the use of > 50%maxTOTPAR defined clinically acceptable pain relief. Number-needed-to-treat (NNT) for one patient to have > 50%maxTOTPAR compared with placebo was used to examine the effectiveness of different single oral doses of tramadol and comparator drugs. Eighteen randomised, double-blind, parallel-group single-dose trials with 3453 patients using categorical pain relief scales allowed the calculation of %maxTOTPAR. The use of > 50%maxTOTPAR was a sensitive measure to discriminate between analgesics. Tramadol and comparator drugs gave significantly more analgesia than placebo. In postsurgical pain tramadol 50, 100 and 150 mg had NNTs for > 50%maxTOTPAR of 7.1 (95% confidence intervals 4.6-18), 4.8 (3.4-8.2) and 2.4 (2.0-3.1), comparable with aspirin 650 mg plus codeine 60 mg (NNT 3.6 (2.5-6.3)) and acetaminophen 650 mg plus propoxyphene 100 mg (NNT 4.0 (3.0-5.7)). With the same dose of drug postsurgical patients had more pain relief than those having dental surgery. Tramadol showed a dose-response for analgesia in both postsurgical and dental pain patients. With the same dose of drug postsurgical pain patients had fewer adverse events than those having dental surgery. Adverse events (headache, nausea, vomiting, dizziness, somnolence) with tramadol 50 mg and 100 mg had a similar incidence to comparator drugs. There was a dose response with tramadol, tending towards higher incidences at higher doses. Single-patient meta-analysis using more than half pain relief provides a sensitive description of the analgesic properties of a drug, and NNT calculations allow comparisons to be made with standard analgesics. Absolute ranking of analgesic performance should be done separately for postsurgical and dental pain.

Acetaminophen↗

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection.

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.

Adult↗

Assay for codeine, morphine and ten potential urinary metabolites by gas chromatography--mass fragmentography.

A mass fragmentography (MF) assay is described for ten potential, minor urinary metabolites of codeine (C) and morphine (M). Samples were hydrolyzed, extracted, derivatized with Tri-Sil Z and analyzed by methane chemical ionization (CI)-MF. The method is sensitive to ca. 0.01 microgram/ml for all compounds with the exception of normorphine (NM) which was difficult to extract with chloroform. The sensitivity of the MF assay for NM was only ca. 0.10 microgram/ml. Various solvent systems were investigated for optimization of extraction efficiency of all metabolites. A separate method for the extraction of NM is reported which utilizes a solid buffer--solvent combination, i.e., potassium carbonate--isopropanol. This latter method provided the best overall recovery of NM (39.0 +/- 3.4%). Gas chromatographic (GC) retention times of C, M and metabolites are reported for three liquid phases (3%) on Gas-Chrom Q (100-120 mesh). Resolution of metabolites (as trisilyl derivatives) was best on Silar-5CP and this phase was used in metabolic studies of C and M. GC resolution was not complete for all compounds; however, selection of specific ions for monitoring by MF provided the required specificity for all compounds except the 6 alpha- and 6 beta-hydroxy isomers. CI spectra for all metabolites are reported. The MF assay was used for urinary analysis of samples from guinea pigs that received single doses of C (15 mg/kg) or M (8 mg/kg). Following C administration 6 alpha- and 6 beta-hydrocodol, 6 alpha, beta-hydromorphol (undifferentiated), HM and M were measured. Following M administration only 6 alpha, beta-hydromorphol was found. The amount of total metabolite as percent dose for each component was calculated as less than 1%.

Animals↗

Simultaneous spectrofluorimetric determination of (acetyl)salicylic acid, codeine and pyridoxine in pharmaceutical preparations using partial least-squares multivariate calibration.

A partial least-squares calibration (PLS) method for the simultaneous spectrofluorimetric determination of salicylic acid (SA), codeine (CO) and pyridoxine (PY) is proposed. The determination of SA, CO, and PY has been carried out in mixtures of up to three components by recording the emission fluorescence spectra between 300 and 500 nm (lambda(exc) = 220 nm). Due to the fact of the strong spectral overlap among the excitation and also among the emission spectra of these compounds, a previous separation should be carried out in order to determine them by conventional spectrofluorimetric methodologies. Here, a full-spectrum multivariate calibration PLS method is developed. The experimental calibration matrix was constructed with 14 samples. The concentration ranges considered were 0.1-2.0 (SA), 0.25-3.0 (CO) and 0.10-2.0 (PY) mg x l(-1). The optimum number of factors was selected by using the cross-validation method. The method also allows the simultaneous determination of acetylsalicylic acid (ASA), CO and PY by previous alkaline hydrolysis of ASA to SA. To check the accuracy of the proposed method, it was applied to the determination of these compounds in synthetic mixtures and in pharmaceuticals.

Analgesics, Opioid↗

A chromatographic method for rapid and simultaneous analysis of codeine phosphate, ephedrine HCl and chlorpheniramine maleate in cough-cold syrup formulation.

The present paper describes a simple, accurate and precise reversed phase HPLC method for rapid and simultaneous quantification of codeine phosphate, ephedrine HCl and chlorpheniramine maleate in a cough-cold syrup formulation. Separations were carried out on a Zorbax XDB C8 column (150 x 4.6 mm ID), 5 microm particle size. A gradient elution system was developed using varying percentages of two mobile phases: methanol-glacial acetic acid-triethylamine (980:15:6 v/v) and water-glacial acetic acid-triethylamine (980:15:6 v/v). The elution of the analytes was achieved in less than 7 min with a flow rate of 1.5 ml/min. Detection was by UV absorbance at a wavelength of 254 nm. Quantification of the components in actual syrup formulations was calculated against the responses of freshly prepared external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control recommended by FDA of the USA.

Chemistry, Pharmaceutical↗

Determination of morphine and codeine in plasma by HPLC following solid phase extraction.

A cheap simple and rapid extraction procedure followed by a UV high performance liquid chromatography (HPLC) assay is described for the simultaneous determination of morphine (M) and codeine (C) in plasma. The method is based on extraction of these opiates from plasma using reversed phase (solid phase) extractions columns followed by HPLC with UV detection at 240 nm. The extraction step provides, respectively, 85 and 80% recovery for M and C. The response of the detection system is linear for both molecules in the studied range from 50 to 750 ng ml-1. No other drugs have been found to interfere with the assay. This method offers a quick, cost effective and reliable procedure for specifically determining M and C, from a small sample volume.

Analgesics, Opioid↗