A Study of Opiate, Opiate Metabolites and Antihistamines in Urine after Consumption of Cold Syrups by LC-MS/MS.

Studying the origin of opiate and/or opiate metabolites in individual urine specimens after consumption of cold syrups is vital for patients, doctors, and law enforcement. A rapid liquid chromatography-tandem mass spectrometry method using "dilute-and-shoot" analysis without the need for extraction, hydrolysis and/or derivatization has been developed and validated. The approach provides linear ranges of 2.5-1000 ng mL-1 for 6-acetylmorphine, codeine, chlorpheniramine, and carbinoxamine, 2.5-800 ng mL-1 for morphine and morphine-3-ß-d-glucuronide, and 2.5-600 ng mL-1 for morphine-6-ß-d-glucuronide and codeine-6-ß-d-glucuronide, with excellent correlation coefficients (R2 > 0.995) and matrix effects (< 5%). Urine samples collected from the ten participants orally administered cold syrups were analyzed. The results concluded that participants consuming codeine-containing cold syrups did not routinely pass urine tests for opiates, and their morphine-codeine concentration ratios (M/C) were not always < 1. In addition, the distribution map of the clinical total concentration of the sum of morphine and codeine against the antihistamines (chlorpheniramine or carbinoxamine) were plotted for discrimination of people who used cold syrups. The 15 real cases have been studied by using M/C rule, cutoff value, and distribution map, further revealing a potential approach to determine opiate metabolite in urine originating from cold syrups.

Trace analysis of three antihistamines in human urine by on-line single drop liquid-liquid-liquid microextraction coupled to sweeping micellar electrokinetic chromatography and its application to pharmacokinetic study.

A rapid and efficient dual preconcentration method of on-line single drop liquid-liquid-liquid microextraction (SD-LLLME) coupled to sweeping micellar electrokinetic chromatography (MEKC) was developed for trace analysis of three antihistamines (mizolastine, chlorpheniramine and pheniramine) in human urine. Three analytes were firstly extracted from donor phase (4 mL urine sample) adjusted to alkaline condition (0.5 M NaOH). The unionized analytes were subsequently extracted into a drop of n-octanol layered over the urine sample, and then into a microdrop of acceptor phase (100 mM H(3)PO(4)) suspended from a capillary inlet. The enriched acceptor phase was on-line injected into capillary with a height difference and then analyzed directly by sweeping MEKC. Good linear relationships were obtained for all analytes in a range of 6.25 × 10(-6) to 2.5 × 10(-4)g/L with correlation coefficients (r) higher than 0.987. The proposed method achieved limits of detections (LOD) varied from 1.2 × 10(-7) to 9.5 × 10(-7)g/L based on a signal-to-noise of 3 (S/N=3) with 751- to 1372-fold increases in detection sensitivity for analytes, and it was successfully applied to the pharmacokinetic study of three antihistamines in human urine after an oral administration. The results demonstrated that this method was a promising combination for the rapid trace analysis of antihistamines in human urine with the advantages of operation simplicity, high enrichment factor and little solvent consumption.

Chemometrics optimization of six antihistamines separations by capillary electrophoresis with electrochemiluminescence detection.

This work expanded the knowledge of the use of chemometric experimental design in optimizing of six antihistamines separations by capillary electrophoresis with electrochemiluminescence detection. Specially, central composite design was employed for optimizing the three critical electrophoretic variables (Tris-H(3)PO(4) buffer concentration, buffer pH value and separation voltage) using the chromatography resolution statistic function (CRS function) as the response variable. The optimum conditions were established from empirical model: 24.2mM Tris-H(3)PO(4) buffer (pH 2.7) with separation voltage of 15.9 kV. Applying theses conditions, the six antihistamines (carbinoxamine, chlorpheniramine, cyproheptadine, doxylamine, diphenhydramine and ephedrine) could be simultaneous separated in less than 22 min. Our results indicate that the chemometrics optimization method can greatly simplify the optimization procedure for multi-component analysis. The proposed method was also validated for linearity, repeatability and sensitivity, and was successfully applied to determine these antihistamine drugs in urine.

Liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine.

A validated method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and several metabolites is presented. The procedure comprises a sample clean-up step, using mixed-mode solid-phase extraction followed by liquid chromatography (LC)-tandem mass spectrometry analysis. Chromatographic separation was achieved using a Sunfire C(8) column eluted with a mixture of formate buffer, methanol, and acetonitrile. The applied LC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated analogues. Validation of the method was performed using 500 microL of urine. The limits of quantification (LOQ) for LSD and 2-oxo-3-hydroxy-LSD were 0.05 and 1 ng/mL, respectively, and ranged, for the other hallucinogens, from 0.5 to 10 ng/mL. Linear and quadratic regression was observed from the LOQ of each compound to 12.5 ng/mL for LSD, 50 ng/mL for 2-oxo-3-hydroxy-LSD and 500 ng/mL for the others (r(2) > 0.99). Precision for the QC samples, spiked at a minimum of two concentrations, was calculated [%CV and %bias < 20% for most of the compounds, except for bufotenine and cathinone (%bias < 24%), and ibogaine (%bias < 30%)]. Extraction was found to be both reproducible and efficient with recoveries > 87% for all the analytes. Furthermore, the processed samples were demonstrated to be stable in the autosampler for at least 24 h. Finally, the validated method was applied to the determination of chlorpheniramine, ketamine, LSD, and psilocin in authentic urine samples.

Postmortem diffusion of drugs from the bladder into femoral venous blood.

We describe significantly elevated drug concentrations in the femoral venous blood due probably to postmortem diffusion from the bladder. A 16-year-old deceased male was found in a shallow ditch in winter. The estimated postmortem interval was 9 days and putrefaction was not advanced. The cardiac chambers contained fluid and coagulated blood and a small amount of buffy coat clots. Diffused hemorrhages were found in the gastric mucosa. The bladder contained approximately 600 ml of clear urine. Gas chromatographic-mass spectrometric analysis of the urine disclosed allylisopropylacetylurea (a fatty acid ureide sedative), diphenhydramine, chlorpheniramine and dihydrocodeine. The cause of death was considered to be drowning due to a drug overdose and cold exposure. The concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine in the femoral venous blood (1.89, 3.27 and 3.30 microg/ml, respectively) were much higher than those in blood from the right cardiac chambers (0.294, 0.237 and 0.240 microg/ml, respectively). Urine concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine were 22.6, 37.3 and 43.1 microg/ml, respectively. The stomach contained negligible amounts of diphenhydramine, free dihydrocodeine and total dihydrocodeine (0.029, 0.018 and 0.024 mg, respectively); concentrations of these drugs in the femoral muscle were 0.270, 0.246 and 0.314 microg/g, respectively. These results indicate that postmortem diffusion of diphenhydramine and dihydrocodeine from the bladder resulted in the elevated concentrations of these drugs in the femoral venous blood. Not only high urinary drug concentrations but also a large volume of urine in the bladder might accelerate the postmortem diffusion.

Determination of the enantiomers of chlorpheniramine and its main monodesmethyl metabolite in urine using achiral-chiral liquid chromatography.

The enantiomers of chlorpheniramine and its monodesmethyl metabolite were determined separately in urine by using a coupled achiral-chiral chromatographic system. The two enantiomers of the studied compound and the internal standard were separated from the biological matrix on a cyanopropyl column and reinjected into a chiral amylose AD column where the two enantiomers were separated and quantified by UV detection. The method was validated for chlorpheniramine and for the metabolite within the range 0-1000 ng/ml. It was also applied in a pilot pharmacokinetic study to samples from a volunteer given 8 mg of racemic chlorpheniramine by mouth.

Metabolism of chlorpheniramine in rat and human by use of stable isotopes.

1. The metabolism of chlorpheniramine (I) was examined in vivo in rats and a human volunteer; in the rats a stable isotope was used. 2. In addition to the unchanged drug (I) and the N-demethylated metabolites (II and III), nine further metabolites were identified in rat urine, four of which were also found in human urine. Chlorpheniramine N-oxide (IV), 3-(p-chlorophenyl)-3-(2-pyridyl) propanol (V), 3-(p-chlorophenyl)-3-(2-pyridyl)-N-acetylaminopropane (VII) and 3-(p-chlorophenyl)-3-(2-pyridyl)-propionic acid (XIII) were identified in rat and human urine. 3. The hydroxylated metabolites of the pyridyl ring of the unchanged drug, II, V and VII, and the glucuronide of XIII were identified only in rat urine. XIII was found in rat urine as long as 6 days after the last dose.

Chlorpheniramine dissolution and relative urinary excretion from commercial products.

Dissolution profiles were determined for seven commercially available nonprescription solid dosage forms containing chlorpheniramine (four sustained release and three immediate release). An in vitro pH change method used to simulate GI transit produced dissolution profiles for some similarly labeled products which were significantly nonequivalent. One product failed to release its chlorpheniramine even when ground in a mortar and pestle in HCl solution, but did release drug in H3PO4 solution. A small (four subjects) relative bioavailability study based on average cumulative excretion of intact drug in urine gave results in parallel with substantially nonequivalent dissolution data for three products.

Clinical pharmacokinetics of chlorpheniramine.

The clinical pharmacokinetics of chlorpheniramine are reviewed. Recent studies have established that the half-life of chlorpheniramine is longer than previously reported. Chlorpheniramine has a serum half-life of approximately 20 hours in adults, and elimination from the body is primarily by metabolism to monodesmethyl and didesmethyl compounds. The half-life is increased in the presence of renal dysfunction and decreased in children. The exact mechanism of the presystemic first-pass elimination and the effects of dose levels on the process presently are unclear. Biopharmaceutical and pharmacokinetic studies after single or multiple doses in humans reveal wide interindividual variations in pharmacokinetics. Age, dialysis, urinary pH and flow influence the elimination kinetics of chlorpheniramine. Attention is brought to major issues that need further clarification to optimize drug therapy with this antihistamine. The use of pharmacokinetic parameters of chlorpheniramine for clinical application is discussed.

Urinary excretion of chlorpheniramine and its metabolites in children.

The pharmacokinetics and urinary excretion of chlorpheniramine were studied in 11 patients, aged 6-16 years, with allergic rhinitis. In these children, chlorpheniramine had a mean elimination half-life of 13.1 +/- 6.6 h, a mean clearance rate of 7.23 +/- 3.16 mL/min/kg, and a mean apparent volume of distribution of 7.0 +/- 2.8 L/kg. Over 48 h, the recovery in urine was as follows: chlorpheniramine, 11.3 +/- 6.7%; demethylchlorpheniramine , 23.3 +/- 11.1%; and didemethylchlorpheniramine , 9.6 +/- 9.4%. Urine flow rate and urine pH were uncontrolled and ranged from 2.2 to 113.3 mL/h and 5.1-7.9, respectively, over the 48-h period. In some children urine flow rate and pH were constant, while in others there was great variability. When drug and metabolite excretion rates versus both urine flow rates and pH values were analyzed by multiple linear regression, the results were significantly better (p less than or equal to 0.05) than when each factor was analyzed independently. The excretion rate of chlorpheniramine and its two demethylated metabolites decreased as urine pH increased and urine flow rate decreased. This information must be considered in future pharmacokinetic studies of this drug.

Isolation and identification of the polar metabolites of chlorpheniramine in the dog.

The metabolites of chlorpheniramine were isolated from dog urine. After daily repeated dosing with chlorpheniramine, [methylene-14C]chlorpheniramine maleate was given as a tracer and urine was collected until less than 1% of the labeled dose was excreted daily. An average of 54% of the oral radioactive dose was recovered in the urine. In addition to the N-demethylated metabolites, one very polar metabolite accounting for about 18% and two less polar metabolites accounting for a total of about 30% of the total urine radioactivity were isolated. Hydrolysis studies of the most polar metabolite indicated that it was a conjugate, though not a glucuronide or sulfate. The metabolite identified after hydrolysis was 3-(p-chlorobenzyl)-3-(2-pyridyl)propionic acid. One of the two less polar metabolites was identified as the corresponding alcohol. The least abundant metabolite could not be identified.

Urinary excretion of chlorpheniramine and pseudoephedrine in humans.

A specific high-pressure liquid chromatographic method for the determination of chlorpheniramine and pseudoephedrine in urine was developed and applied in a urinary excretion study of normal healthy subjects who received a sustained-release dosage form contianing 8 mgof chlorpheniramine maleate and 120 mg of pseudoephedrine hydrochloride. Five subjects received one dose on Day 1, followed by multiple dosing every 12 hr for 7 days without ammonium chloride administration. Four subjects received one dose of the sustained-release dosage form together with ammonium chloride. Urine samples were collected during the 1st day and at steady state. The method is specific and simultaneously determines choorpheniramine, two metabolites (mono- and di-desmethylchlorpheniramine), pseudoephedrine, and norpseudoephedrine. The assay recovery was less than 97% (0.06-3 microgram/ml) for chlorpheniramine maleate and less than 98% (1.5-75 microgram/ml) for pseudoephedrine hydrochloride. Excretion of chlorpheniramine and its two metabolites in urine was enhanced after ammonium chloride administration. At steady state, a change in urine pH from 5.69 to 6.46 resulted in more than a 25% decrease in chlorpheniramine and monodesmethylchlorpheniramine excretion. In spite of expected changes in its biological half-life, the overall amount of unchanged pseudoephedrine excreted in urine was not affected by urine pH, presumably because it is primarily excreted in urine as intact drug.

Chlorpheniramine. I. Rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine by high-performance liquid chromatography.

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.

Simultaneous GLC determination of phenylpropanolamine and chlorpheniramine in urine using a nitrogen selective detector.

A simple, rapid, and sensitive simultaneous quantitative determination of phenylpropanolamine and chlorpheniramine in human urine by GLC, using a nitrogen specific detector, is described. After alkaline extraction from urine, phenylpropanolamine and chlorpheniramine are analyzed directly by GLC, without a derivatization step. Promethazine was used as the internal standard. The total assay time is less than 30 min. The method is useful in studies of pharmacokinetic and pharmacological interactions of drug combinations.