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Radioimmunoassay of Buprenorphine in Urine: Studies in Patients and in a Drug Clinic

C.W. Hand, Karen E. Ryan, Sujata K. Dutt, and R.A. Moore*
DPC European Research Institute, Station Lane, Witney, Oxfordshire OX8 6AN, United Kingdom

J.O'Connor and Dymphna Talbot
Drug Centre and Toxicology Department, Jervis Street Hospital, Dublin 1, Ireland

H.J. McQuay
Oxford Regional Pain Relief Unit, Abingdon Hospital, Abingdon, Oxford, United Kingdom


A radioimmunoassay kit (DPC buprenorphine double antibody) was evaluated with clinical samples and samples from a drug clinic. Urine samples were collected over a 2-day period from 5 hospital in-patients receiving sublingual buprenorphine, 400 to 2000 µg/day, for the relief of chronic pain. Samples were measured before and after enzymatic hydrolysis. Urine buprenorphine concentrations were measurable at all doses studied (minimum value 5.6 ng/mL) and were greater with larger doses. The increase in concentration after hydrolysis averaged 49% and was similar for all doses studied. The authors conclude that the method has extensive cross-reactivity with glucuronides of buprenorphine and its metabolites and that samples may be analysed without prior hydrolysis. The prevalence of buprenorphine use in 97 patients attending a drug clinic was also studied. Sixty (62%) had measurable urinary buprenorphine concentrations of 1 ng/mL or more by direct assay. The buprenorphine users were significantly younger and reported significantly greater use of opiates than nonusers.


Buprenorphine is a powerful agonist analgesic and is effective in treating many forms of pain (1). Its kinetics are well understood (2). Despite influential findings of low addiction potential (3) and the suggestion that it may be useful in the management of opiate addicts (4), there have been reports of misuse (5-7). Indeed, it has been estimated that 50% of hardcore drug users in New Zealand use buprenorphine (6), although this may reflect the fact that buprenorphine is more easily obtained than are more strictly controlled drugs. Impressions of the prevalence of buprenorphine use, however, have not been validated by screening.A joint study by the Medico-Social Research Board and the National Drug Advisory and Treatment Centre5 or as it is commonly referred to, the Jervis Street Drug Centre, of patients attending the Drug Centre from 1979-1983 showed that during this five year period there had been a six fold increase in the number of persons making their first contact with the Centre and a four-fold increase in re-contacts. Males exceeded females by a ratio of three to one and the majority of attenders, approximately 60%, were aged under 25 years. Opiates were the principal drugs of misuse and in particular, heroin. Between 1979 and 1983 the numbers contacting Jervis Street Drug Centre for the first time for treatment of opiate misuse rose eight-fold from 56 to 451. This increase, together with evidence from the Garda Drug Squad of a dramatic increase in the number of persons charged for heroin-related offences and of seizures of the drug, confirmed that there had been an "opiate epidemic" in Dublin between 1979 and 1983.6 When the attendance at the Jervis Street Drug Centre was analysed for area of residence the attendance rates were highest from north and south central Dublin. The Medico-Social Research Board and the Jervis Street Drug Centre continued their collaboration in a study of patients who had received treatment at the Centre in 1984. This paper reports on the characteristics of the 1984 attenders making some comparisons with those of previous years and also comments on information collected for the first time in 1984. More recent information, as it is available, for 1985 and 1986 is also presented.

The plasma kinetics of buprenorphine after intravenous, intramuscular, and sublingual administration have been well studied over a number of years (1,2,8). Excretion of buprenorphine and its metabolites in urine have been less well studied. Using GC/MS, Conet et al. (9) reported urinary excretion of about 2, 13, and 12% of subcutaneous, sublingual and oral doses respectively. No unconjugated buprenorphine was found, and the urine excretion was as glucuronide conjugates of buprenorphine and N-dealkylbuprenorphine with small amounts of unconjugated N-dealkylbuprenorphine. In a single subject, Blom et al. (10) found small amounts of buprenorphine in the unconjugated form.

Immunoassay is the preferred method for screening urine for buprenorphine because chromatographic methods generally lack sensitivity (11). This study was designed to examine a new urinary buprenorphine radioimmunoassay kit, to determin urinary buprenorphine levels in patients taking the drug for relief of chronic pain, and to investigate the effects of hydrolysis on observed levels.

A small pilot study employing the assay among patients in the Dublin Drug Centre in early 1987 appeared to indicate a high incidence of buprenorphine use (unpublished data). The study was therefore extended to survey the drug centre population for the prevalence of buprenorphine use.

Materials and Methods

Assay validation: The DPC buprenorphine in urine double antibody kit was used as supplied by the manufacturer (EURO/DPC Ltd.). N-Dealkylbuprenorphine and etorphine were gifts from Reckitt & Colman Pharmaceuticals Ltd. Controls containing 0, 5, and 15 ng/mL of buprenorphine (CONDOA-T, EURO/DPC) were included in all assays. For testing cross-reactions in the assay, drugs were made up in concentrations of 100,000 ng/mL in drug-free human urine. N-Dealkylbuprenorphine and etorphine (and other drugs that gave cross-reaction at 100,000 ng/mL) were diluted until values on the standard curve were obtained; buprenorphine-3-glucuronide and N-dealkylbuprenorphine glucuronide were not available for testing.

Sample hydrolysis: In order to determine whether buprenorphine-3-glucuronide, N-dealkylbuprenorphine glucuronide (and possibly buprenorphine-3-sulfate) are detected by the assay, samples from chronic pain patients were subjected to enzymatic hydrolysis. For hydrolysis, patient samples (100 uL) were diluted 1:1 with 0.01 mol/L citrate/phosphate buffer pH 5.0 containing 200 units/mL and 80 units/mL respectively of β-glucuronidase (EC, type H1 from Helix pomatia, Sigma Chemical Co.) and Aryl sulfatase (EC, type V, from Patella vulgata, Sigma).

Drug-free human urine: Urine samples were collected from 50 volunteers known not to be taking any drugs or medications.

Chronic pain patients: Five patients (3 female) attending the pain relief clinic were studied. All were taking buprenorphine sublingually for relief of chronic pain; none had any clinically apparent renal or hepatic disorder. The buprenorphine dose was 400 to 2000 µg in divided sublingual doses, and the dose of buprenorphine had not changed for at least 1 week before sample collection. Each patient provided urine samples over a 2-day period while in hospital; a total of 25 samples were collected. Urine samples were frozen without preservative until analysed.

Drug clinic patients: In August 1987, patients attending the Jervis Street Drug Centre gave the usual urine sample, and a form was completed by the physician noting the age and sex of the patient, what drugs they were accustomed to using, and whether they were on methadone maintenance. Urine samples were screened for the presence of drugs by thin-layer chromatography (TLC). A buprenorphine test was taken as positive if the measured concentration (on duplicate samples) was equal to or greater than 1.0 ng/mL. Actual concentrations were recorded.

Statistical differences between the ages of patients and the pattern of drug use for those who were positive or negative for buprenorphine in the urine were tested with the Mann Whitney U test or the chi-square test (with Yates correction). In all cases two-tailed tests of statistical significance were used. Mean values and standard error of the mean are reported.


The buprenorphine assay gave a standard curve between 1 and 25 ng/mL (an example assay is shown in Table I). Interassay precision was calculated from 20 individual assays; the zero control always read below 0.1 ng/mL (by extrapolation). The mean value obtained for the 5-ng/mL control was 4.8 ng/mL (coefficient of variation (CV), 6.3%) and that for the 15-ng/mL control was 15.2 ng/mL (CV 6.9%).

A wide variety of drugs (Appendix A) was assayed at a concentration of 100,000 ng/mL and found not to cross-react. Eight drugs gave detectable readings in the assay. N-Dealkylbuprenorphine gave a cross-reaction of 98% when tested at 10 ng/mL, and etorphine gave a cross-reaction of 1.8% when tested at 100 ng/mL. Diprenorphine showed a maximum cross-reaction of less than 0.1%; prednisolone; hydromorphone, dextromethorphan, N-dealkyletorphine, and nalbuphine displayed less than 0.05% cross-reaction.

Positive urine samples diluted with zero calibrator gave results that were parallel with the standard curve.

Drug-free human urine: results for 50 drug-free human volunteers were all below 1.0 ng/mL; the maximum observed concentration by linear extrapolation of the standard curve was below 0.1 ng/mL.

Chronic pain patients. Results for individual patients and samples are given in Table II. The lowest value found in a single urine by direct assay was 5.6ng/mL. There were marked variations in urine concentrations and in concentration increases after hydrolysis (no dilution effect was apparent when diluting samples in hydrolysis buffer without enzymes). Despite this, the urinary concentration of buprenorphine was higher with a higher daily dose of the drug, both for the direct assay and after hydrolysis. The mean increase after hydrolysis was 49% for all 25 samples, but the range was great - between about 13 and 190% (median about 30%).

Drug clinic patients: Complete records for 94 of the 97 patients studied were obtained; all 97 results were included in the buprenorphine analysis, and 94 were used for demographic analysis.

Measurable urinary buprenorphine concentrations (1.0 ng/mL or greater) were found in 60 (62%) of the 97 patients, and the number of samples in different concentration ranges are shown in Table III. Eleven patients had results between 1.0 and 1.9 ng/mL, and of these, only two had urine drug test positive for other drugs (one each for opiates and benzodiazepines).

The demographic data on the patients with buprenorphine-positive or negative urine samples is given in Table IV. Those in the buprenorphine-positive group were significantly younger (p<0.025, Mann Whitney U test), but there was no difference in sex ration. Buprenorphine users reported the use of nearly twice as many drugs as those with negative urine test (p<0.001, Mann Whitney U test), and this extra use was limited to opiate agonists (p>0.005) and buprenorphine (P<0.025, chi-square test). The proportion of patients on methadone maintenance or who had positive urine tests for drugs other than buprenorphine or methadone (predominantly benzodiazepines and opiates) were the same in both groups.


Buprenorphine is a safe and effective analgesic, and its abuse potential is usually considered lower than that of other opiate agonists (3) because of its ability as an antagonist to block the subjective effects of morphine and because it does not produce physical dependence. The agonist properties of buprenorphine, on the other hand, have suppressed heroin self-administration in addicts (4) and have induced buprenorphine self-administration in monkeys (12). Lethal overdose appears to be impossible, however (14).

After sublingual administration, buprenorphine plasma concentration rise slowly and are maintained at a low concentration (1-3 ng/mL) for some hours (14). Buprenorphine has a long half-life of about 8 h (1,2), and the N-dealkyl metabolite appears to have an even slower elimination (8). This accords with the appearance of buprenorphine and its glucuronide in urine in 1-2 days, and N-dealkylbuprenorphine and its glucuronide of 1-4 days (9,10).

For detection of buprenorphine in urine a test must be sensitive and must measure not only buprenorphine but also its major metabolites. The DPC double antibody buprenorphine assay has a calibration range of 1 to 25 ng/mL and gives low coefficients of variation at 5 and 15 ng/mL; apparent buprenorphine concentrations in the urine of drug-free individuals were below 0.1 ng/mL. The assay does not cross-react with a wide variety of drugs, but does cross-react significantly with a one major metabolite, N-dealkylbuprenorphine. The assay is considered to be sufficiently specific for use as a urine screening test for buprenorphine.

The other major urinary excretion products of buprenorphine, buprenorphine-3-glucuronide and N-dealkylbuprenorphine glucuronide, were not available for cross-reaction testing. Buprenorphine is excreted by humans almost exclusively in conjugated form (9, 10). If the assay detects glucuronides of buprenorphine and N-dealkylbuprenorphine, enzymatic hydrolysis would be expected to make only a small difference to apparent concentrations; if these metabolites are not detected, then hydrolysis would be expected to increase considerably the apparent urine concentration (by some 4 to 9 times). In the 5 patients studied here over a daily dosage of 400 to 2000 µg buprenorphine, the mean increase in observed concentration after hydrolysis was between 29 and 75%. The implication is that cross-reaction to glucuronides was extensive.

Urine buprenorphine values in patients administered sublingual buprenorphine were all above the lowest calibrator in the assay.

Urine buprenorphine concentrations were variable, but generally increased with dosage. The minimum urine concentration found was about 5 ng/mL by direct assay in a patient receiving only 400 µg/day. Mean systemic availability of sublingual buprenorphine is only about 65% (14), and so 400 µg sublingually is equivalent to 250 µg given parenterally. This is less than a standard injected analgesic dose of buprenorphine (and equivalent to about 7mg intravenous morphine). Measurement of urine buprenorphine concentrations by radioimmunoassay should therefore detect any illicit use of buprenorphine, although the data presented here do not indicate how long after a dose buprenorphine may be detected in urine.

In screening 97 patients from a drug clinic during a 1-month period, the buprenorphine test indicated that 62% of the samples had values greater than 1 ng/mL. This raises the question of the appropriate cut-off value for the presence of buprenorphine in urine. The extreme position of using 1 ng/mL, the concentration of the lowest calibrator, was taken here. This is supported by the specificity of the method, by the values of less than 1 ng/mL obtained in drug-free individuals, and by the similar frequency of methadone use and urine tests positive for other drugs in the two patient groups (Table IV). As drug testing guidelines evolve, different considerations may determine other values. The proportion of positive test at a cut-off of 1 ng/mL was 62%, and was 51%, 46%, 42% and 39% at 2, 3, 4 and 5 ng/mL respectively. A reliable confirmation method for buprenorphine in urine is required.

In this test, buprenorphine users were significantly different from the group testing negative. They were younger, they had experimented with more drugs (specifically opiates), and 56% admitted using buprenorphine (compared with only 17% in the buprenorphine-negative group). This pattern of buprenorphine use by hard-core drug users is similar to that reported anecdotally in New Zealand and Edinburgh (6,7).

A major reason for use of buprenorphine by experienced opiate addicts attending drug clinics, apart form its opiate agonist properties and availability, has been the virtual impossibility of its detection in urine samples. The extremely low urine concentration of buprenorphine and its metabolites make detection by TLC highly unlikely, while HPLC also lacks sensitivity (11) and GC/MS is time-consuming and expensive. Implementation of drug clinic screening for buprenorphine and its metabolites by immunological methods should be an effective means of reducing the attractiveness of buprenorphine use among opiate addicts.


We wish to tank Sister Dawn Carroll for organising the collection of samples and the ward staff of the Oxford Regional Pain Unit of their assistance.

Appendix A

Compounds that do not cross-react with the buprenorphine assay*

Acetaminophen (Paracetamol)
Acetylsalicylic acid
Allylcyclopentyl barbituric acid
Barbituric acid
Cotinine (nicotine metabolite)
5,5-Diallyl barbituric acid
Ethyl morphine
Lysergic acid

* These compounds were undetectable in the buprenorphine assay at a concentration of 100,000 ng/mL. Eight other compounds were detectable by the buprenorphine RIA. Of these 6 had cross-reaction of less than 0.1% (see text).


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