Radioimmunoassay of carteolol in human plasma

A radioimmunoassay for the direct measurement of carteolol, a new beta-adrenoreceptor blocker, in human plasma was developed. Carteolol was acylated to form O-glutarylcarteolol, which was conjugated to bovine serum albumin to provide the immunogen. Antibody to carteolol was raised in New Zealand albino rabbits. The tracer was the radioiodinated derivative of O-glutarylcarteolol-tyrosine methyl ester conjugate. The method is highly sensitive, with a lower quantifiable concentration of approximately 0.4 ng of carteolol/ml using 0.1 ml of plasma, and has good specificity, with the major metabolite (8-hydroxycarteolol) showing only 0.2% cross-reactivity. It is reproducible, with relative standard deviations from triplicate standard curves being mostly within +/- 8%. The method is currently being used to monitor carteolol levels in clinical samples.     

Radioimmunoassay of glipizide in human plasma

Summary A simple, sensitive radioimmunoassay has been developed for the direct determination of glipizide in human plasma. Antisera raised in rabbits immunized with a glipizide analogue conjugated to bovine serum albumin were highly specific, the two main metabolites, 3,cis-hydroxycyclohexyl derivative and 4,trans-hydroxycyclohexyl derivative, having cross reactivities of 0.73% and 1.66%, respectively. The method can measure amounts as small as 1 ng/ml. The intra- and inter-assay coefficients of variation lay between 2.98–5.79% and 2.35–8.66%, respectively. The mean recovery of glipizide added to plasma was 99–105% over the range 1–500 ng/ml. The method was employed to determine plasma levels in six subjects after administration of a 5 mg tablet of glipizide. The results were in accordance with those found after administration of the same dose of radiolabelled glipizide to two other subjects.     

Radioimmunoassay of flurazepam in human plasma

A radioimmunoassay for the determination of the hypnotic flurazepam in plasma was developed. Plasma levels of intact flurazepam were measured following oral administration of therapeutic doses to humans. The procedure employs an antiserum obtained from a rabbit immunized with 3-hemisuccinyloxyfluorazepam covalently coupled to bovine serum albumin and tritium-labeled flurazepam as the radioligand. Assay specificity was achieved by chromatographic isolation of flurazepam from plasma extracts on Sephadex LH-20 prior to analysis. The method has a sensitivity limit of 0.1 ng of flurazepam/ml using a 1-ml plasma sample, and the intra- and interassay coefficients of variation did not exceed 9% over a range of 0.1-5 ng/ml. In eight subjects who received a single 30-mg dose of flurazepam, peak plasma concentrations of 0.5-3.0 ng of intact drug/ml were reached after 0.5-1 hr, except in one subject where the peak occurred at 4 hr. The plasma concentrations of flurazepam declined with a harmonic mean apparent half-life of 2.3 hr.     

Radioimmunoassay for prochlorperazine in human plasma

A new sensitive, specific, and rapid radioimmunoassay procedure for the determination of plasma concentrations of the antiemetic drug prochlorperazine is described. The assay enables the quantitation of 31 pg of the drug in 200 microliters of plasma with a coefficient of variation of approximately 2%. Except for N-desmethylprochlorperazine, the antiserum did not cross-react with the available metabolites tested. Also there was no cross-reactivity with the tricyclic antidepressants and antianxiety agents commonly co-administered with the drug. The method is suitable for single-dose pharmacokinetic and bioavailability studies. It should be adequate for the therapeutic monitoring of the drug in patients.     

Radioimmunoassay of acipimox in human plasma and urine

A radioimmunoassay was developed for the determination of acipimox (5-methylpyrazinecarboxylic acid 4-oxide) in human plasma and urine. Acipimox was conjugated to bovine serum albumin through a spacer with 4 carbon atoms, and repeatedly injected into rabbits. Antisera raised in these animals were highly specific and virtually no cross-reaction was observed with 5-methylpyrazinecarboxylic acid and nicotinic acid. Despite the low specific activity of the labelled antigen used, concentrations of acipimox in human plasma and urine as low as 40 ng/ml could be determined. The intra- and inter-assay coefficients of variation ranged between 4.32-6.25% and 6.19-11.55% respectively, and mean recovery of the compound spiked to plasma was 100-103%. The method was applied to determine plasma levels and urinary excretion of acipimox after oral doses of 150 mg and 250 mg to four volunteers.     

Radioimmunoassay for trifluoperazine in human plasma.

1 A new sensitive and rapid radioimmunoassay procedure for the determination of the plasma concentrations of the neuroleptic drug trifluoperazine is described. 2 The antiserum developed for trifluoperazine cross-reacted with N-desmethyltrifluoperazine and 7-hydroxytrifluoperazine to the extent of 26 and 24% respectively but its cross-reactivity with commonly co-administered tricyclic antidepressants and antianxiety agents tested was negligible. 3 The assay, based on the above antiserum, enabled the quantitation of 50 pg of the drug in 200 microliters of plasma with a coefficient of variation of about 2% and therefore should be applicable for singly dose pharmacokinetic and bioavailability studies. It should be applicable to therapeutic monitoring of the drug in patients.     

Radioimmunoassay for perphenazine in human plasma.

1 A new sensitive, specific and rapid radioimmunoassay procedure for the determination of plasma concentrations of the neuroleptic drug perphenazine is described. 2 The antiserum developed for perphenazine did not cross-react with most of the major metabolites of perphenazine nor the tricyclic antidepressants and antianxiety agents commonly co-administered with the drug. 3 The assay, based on the above antiserum, enabled the quantitation of 50 pg of the drug in 200 microliters of plasma with a coefficient of variation of about 8% and therefore should be applicable for single dose pharmacokinetic studies, as well as therapeutic monitoring of the drug in patients.     

Radioimmunoassay for danazol in human and monkey plasma.

A sensitive method is described for the radioimmunoassay of danazol in monkey and human plasma. Antiserum was developed in rabbits, and a second antibody was used to separate bound from free danazol. The radioimmunoassay was specific for danazol, and the limit of detection ranged from 1.4 to 2.8 ng/ml. Exogeneous danazol could be quantitated accurately in both monkey and human plasma. The radioimmunoassay results agreed with values obtained by inverse isotope dilution after intravenous administration of 14C-danazol to monkeys. The assay was used successfully to measure danazol in plasma from human volunteers receiving 200 mg of danazol.     

Inhibitors of cytochrome P450 differentially modify discriminative-stimulus and antinociceptive effects of hydrocodone and hydromorphone in rhesus monkeys.

The present study was conducted to investigate the role of cytochrome P450 in the discriminative-stimulus and antinociceptive effects of hydrocodone (HC) and hydromorphone (HM) in rhesus monkeys. In morphine-deprived monkeys, morphine dose-dependently reversed naltrexone-lever responding, an effect also produced by HC and HM. HC and HM also produced antinociception in a warm-water tail withdrawal procedure. Budipine and naltrexone shifted the dose-effect curves for the discriminative-stimulus effects of HC and HM to the right. In contrast, naltrexone, but not budipine (10.0 mg/kg) or quinidine (10.0 mg/kg), dose-dependently antagonized the antinociceptive effects of HC. Budipine and quinidine decreased the concentration of HM in plasma without significantly affecting the levels of HC, suggesting that these CYP2D6 inhibitors decreased the conversion of HC HM. Thus, some behavioral effects of HC are not modified by a marked inhibition of CYP2D6, suggesting that these effects of HC are not due to its conversion to HM but, rather, that both HC and HM act directly on mu receptors.     

Relationship between the concentration of hydrocodone and its conversion to hydromorphone in chronic pain patients using urinary excretion data

Hydrocodone in combination with acetaminophen is commonly used to control moderate pain and is metabolized by cytochrome P4502D6 to form the active metabolite, hydromorphone. The purpose of this study was to determine the metabolic relationship and variability between hydrocodone and its conversion to hydromorphone using urinary excretion data from chronic pain patients. Liquid chromatography-tandem mass spectrometry was used to quantitate hydrocodone and hydromorphone concentrations in urine specimens. The first visits of 25,200 subjects who took hydrocodone and not hydromorphone and had measurable concentrations were included in this study. The geometric mean (95% confidence index) of hydrocodone and hydromorphone urine concentrations were 1.39 (1.37-1.41) mg per gram of creatinine and 0.224 (0.221-0.227) mg per gram of creatinine, respectively. The log of creatinine-corrected hydromorphone versus the log of creatinine-corrected hydrocodone showed a positive relationship (R(2) = 0.20), with 60-fold variability between subjects. The plot of the log of the metabolic ratio ([hydromorphone] divided by [hydrocodone]) versus the log of creatinine-corrected hydrocodone had a coefficient of determination of R(2) = 0.42, with 125-fold variability between subjects. Ultra-rapid metabolizers represented 0.6% of the population, whereas 4% were poor metabolizers. Within-subject variability for the excretion of hydrocodone in urine was 23-fold, whereas between-subject variability was 134-fold. Hydrocodone and hydromorphone urine concentrations showed great variability within and between subjects.     

Radioimmunoassay of pyridostigmine in plasma and tissues

A sensitive and specific radioimmunoassay is reported for the cholinergic agent, pyridostigmine. Antibodies were raised in rabbits against a conjugate of pyridostigmine and bovine serum albumin. The assay can detect as little as 2.5 ng/ml of drug directly in plasma and tissue homogenates. Structurally similar compounds and major metabolites are not recognized by the antibody. The specificity of the antibody has been confirmed by utilizing high pressure liquid chromatography. Plasma concentration-time profiles and tissue distribution of the drug were determined by this method in rat after intramuscular administration of pyridostigmine.     

GC-MS quantitation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in blood

A method is described for the simultaneous analysis of seven opiates, codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone, in blood samples by gas chromatography-mass spectrometry (GC-MS). One milliliter of blood is combined with an internal standard mixture containing 200 ng of each of the seven deuterated opiates. Two milliliters of acetonitrile is added to precipitate the proteins and cellular material. After centrifugation, the clear supernatant is removed, and the acetonitrile is evaporated. The remaining aqueous portion is adjusted to pH 9 with sodium bicarbonate buffer, and the drugs are extracted into chloroform/ trifluoroethanol (10:1). The organic extractant is transferred and dried under nitrogen. The residue is reconstituted in dilute hydrochloric acid and washed consecutively with hexane and chloroform. The purified aqueous portion is adjusted to pH 9 with bicarbonate buffer, and the drugs are again extracted into chloroform/trifluoroethanol (10:1). The organic portion is removed from the aqueous fraction and dried under nitrogen. The residue is consecutively derivatized with methoxyamine and propionic anhydride using pyridine as a catalyst. The ketone groups on hydrocodone, hydromorphone, oxycodone, and oxymorphone are converted to methoximes. Hydroxyl groups present at the O(3) and O(6) positions of codeine, morphine, 6-acetylmorphine, hydromorphone, and oxymorphone are converted to their respective propionyl esters. After a post-derivatization purification step, the extracts are analyzed by full scan GC-MS using electron impact ionization. The method is linear to at least 2000 ng/mL. Day-to-day precision (N = 15) at 500 ng/mL and 75 ng/mL were less than 10% for all seven targeted opiates. Extraction efficiencies at these two concentrations ranged from 50% to 68%. For each opiate, the limit of quantitation was 10 ng/mL, and the limit of detection was 2 ng/mL.     

High-pressure liquid chromatographic method for the determination of hydromorphone in human plasma with electrochemical detection

A high-pressure liquid chromatographic method with internal analogue standardization for the determination of hydromorphone (Dilaudid) in human plasma is described. The method involves extraction from plasma with chloroform and quantification with an electrochemical detector after high-pressure liquid chromatographic separation on an ion-pair column. Using 3 ml plasma the detection limit is about 50 pg/ml; in the routine procedure the limit of determination can be fixed at about 250 pg/ml with a correlation coefficient of about 10%. The method is selective. In case of extractions from blank plasma samples interfering peaks are not observed.     

GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine.

A procedure for the simultaneous confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine specimens by gas chromatography-mass spectrometry (GC-MS) is described. After the addition of nalorphine and naltrexone as the two internal standards, the urine is hydrolyzed overnight with beta-glucuronidase from E. coli. The urine is adjusted to pH 9 and extracted with 8% trifluoroethanol in methylene dichloride. After evaporating the organic, the residue is sequentially derivatized with 2% methoxyamine in pyridine, then with propionic anhydride. The ketone groups on hydrocodone, hydromorphone, oxycodone, oxymorphone, and naltrexone are converted to their respective methoximes. Available hydroxyl groups on the O3 and O6 positions are converted to propionic esters. After a brief purification step, the extracts are analyzed by GC-MS using full scan electron impact ionization. Nalorphine is used as the internal standard for codeine, morphine, and 6-acetylmorphine; naltrexone is used as the internal standard for the 6-keto-opioids. The method is linear to 2000 ng/mL for the 6-keto-opioids and to 5000 ng/mL for the others. The limit of quantitation is 25 ng/mL in hydrolyzed urine. Day-to-day precision at 300 and 1500 ng/mL ranged between 6 and 10.9%. The coefficients of variation for 6-acetylmorphine were 12% at both 30 and 150 ng/mL. A list of 38 other basic drugs or metabolites detected by this method is tabulated.     

Simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine in urine, serum, plasma, whole blood, and meconium by LC-MS-MS

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous analysis of six major opiates in urine, serum, plasma, whole blood, and meconium is described. The six opiates included are codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine (6-AM). The method was compared to an in-house gas chromatography (GC)-MS method and an LC-MS-MS method performed by another laboratory. The sample preparation time was decreased by eliminating the glucuronide hydrolysis and derivatization required for GC-MS analysis, as well as by adapting the solid-phase extraction to elute directly into autosampler vials. These improvements illustrate the advantages of an LC-MS-MS method over a GC-MS method for opiates. The structural similarity of these six opiates and others in the opiate class causes a high potential for interference and false-positive results. Twelve opiate analogues and metabolites were evaluated for interference. The potential for interference was reduced by altering the MRM transitions chosen for the six opiates. The increased specificity of LC-MS-MS decreased the interference rate in urine to 3.9% compared to 13.6% on the in-house GC-MS method. The rate of positivity for 6-AM in meconium is described for the first time. In urine, 11.0% of morphine positive specimens were also positive for 6-AM compared to 8.3% in serum/plasma and 0.9% in meconium. Although 6-AM is infrequent in meconium, it provides a definitive proof of illegal heroin abuse by the pregnant mother. This method has been routinely used in our laboratory over the last 6 months on more than 1500 patient specimens.     

Identification of hydrocodone in human urine following controlled codeine administration

Allegations of illicit hydrocodone use have been made against individuals who were taking physician-prescribed oral codeine but denied hydrocodone use. Drug detection was based on positive urine opiate immunoassay results with subsequent confirmation of hydrocodone by gas chromatography-mass spectrometry (GC-MS). In these cases, low concentrations of hydrocodone (approximately 100 ng/mL) were detected in urine specimens containing high concentrations of codeine (> 5000 ng/mL). Although hydrocodone has been reported to be a minor metabolite of codeine in humans, there has been little study of this unusual metabolic pathway. We investigated the occurrence of hydrocodone excretion in urine specimens of subjects who were administered codeine. In a controlled study, two African-American and three Caucasian male subjects were orally administered 60 mg/70 kg/day and 120 mg/70 kg/day of codeine sulfate on separate days. Urine specimens were collected prior to and for approximately 30-40 h following drug administration. In a second case study, a postoperative patient self-administered 960 mg/day (240 mg four times per day) of physician-prescribed oral codeine phosphate, and urine specimens were collected on the third day of the dosing regimen. Samples from both studies were extracted on copolymeric solid-phase columns and analyzed by GC-MS. In the controlled study, codeine was detected in the first post-drug-administration specimen from all subjects. Peak concentrations appeared at 2-5 h and ranged from 1475 to 61,695 ng/mL. Codeine was detected at concentrations above the 10-ng/mL limit of quantitation for the assay throughout the 40-h collection period. Hydrocodone was initially detected at 6-11 h following codeine administration and peaked at 10-18 h (32-135 ng/mL). Detection times for hydrocodone following oral codeine administration ranged from 6 h to the end of the collection period. Confirmation of hydrocodone in a urine specimen was always accompanied by codeine detection. Codeine and hydrocodone were detected in all specimens collected from the postoperative patient, and concentrations ranged from 2099 to 4020 and 47 to 129 ng/mL, respectively. Analyses of the codeine formulations administered to subjects revealed no hydrocodone present at the limit of detection of the assay (10 ng/mL). These data confirm that hydrocodone can be produced as a minor metabolite of codeine in humans and may be excreted in urine at concentrations as high as 11% of parent drug concentration. Consequently, the detection of minor amounts of hydrocodone in urine containing high concentrations of codeine should not be interpreted as evidence of hydrocodone abuse.