ANALYSIS OF BENZODIAZEPINES IN WHOLE BLOOD, PLASMA AND URINE BY 125-1 RADIOIMMUNOASSAY C. W.Hand. Mary Rutterford, R. F. Smith and R. A. Moore DPC-European Research Institute, Witney, Oxfordshire 0X8 6AN Running title: Benzodiazepines RIA Correspondence to: Dr. R.A. Moore DPC European Research Institute 31 Station Lane Witney Oxfordshire 0X8 6AN SUMMARY Two benzodiazepine RIA kits (DPC Double Antibody) formulated for use with either serum or urine samples respectively were used to investigate benzodiazepine concentrations in whole blood, plasma and urine. Both kits use oxazepam calibrators ranging from 10 to 1000 ng/ml in an appropriate matrix and have broad specificity for the drug class. Fifty-three paired samples of haemolysed whole blood and plasma were analysed with the serum kit: 26 pairs were from drug-free volunteers and all blood and plasma samples gave undetectable results. Twenty-seven paired samples were from patients receiving benzodiazepines and of these 26 plasma and 25 blood samples were positive. The average plasma/blood ratios for samples from patients taking a single of benzodiazepine were: for diazepam (5, 10 or 20 mg) 2.1, n=7; for temazepam (10, 20, 40 or 60 mg) 2.3, n=12; and for nitrazepam (5, 10 and 15 mg) 2.0, n=3. The urine kit was compared to another commercially available assay (Roche Abuscreen) using 41 positive (patient urines) and 39 negative (volunteer) samples. There was 100% agreement between the two methods relative to a 100 ng/ml oxazepam cut-off. The DPC kit was also used to detect benzodiazepines in urine after a single, low (1 mg) therapeutic dose of either flunitrazepam (n=5) and lorazepam (n=l). After both drugs, benzodiazepines were detectable for at least 12 hours after the dose. Key Words: benzodiazepines, radioimmunoassay, blood INTRODUCTION Two RIA kits formulated for the detection of benzodiazepines in urine or serum were used to investigate concentrations of benzodiazepines in urine, plasma and whole blood from patients taking benzodiazepines or from drug-free volunteers. The serum kit was used to compare the concentration of benzodiazepines in paired samples of whole blood and plasma in patients taking benzodiazepines as part of multi-drug therapy for the relief of chronic pain. Control samples of paired whole blood and plasma were analysed from drug-free volunteers. 893
The urine kit was compared to another commercially available RIA for benzodiazepines and was also used to examine the concentrations of benzodiazepines detectable in urine after low doses of lorazepam and flunitrazepam. METHODS Radioimmunoassay Both RIA kits were used as supplied by the manufacturer (EURO/DPC Ltd, Witney UK). The assays follow basically similar procedures with the exception of sample size. The RIA for urine uses samples of 50 ul; the serum (plasma & whole blood) assay uses a 25 ul sample. Standard, control or sample were mixed with 100 ul ^251-benzodiazepine and 100 ul antiserum solution. After a 60 minute incubation at room temperature bound and free radioactivity were separated by the addition of 1 ml of cold precipitating solution (donkey anti-sheep/goat in PEG/saline) and centrifugation. Tubes were decanted using a foam rack and the pellets counted for 1 minute in a multi-head gamma counter (DPC Gambyt). Sample concentrations were calculated automatically from a logit-log transformation of the standard data. Specificity Cross-reactivity of both RIA kits was assessed with 18 different benzodiazepines at concentrations of 50, 100, 500, 1000 and 10000 ng/ml in drug-free urine or serum. Percentage cross-reactivity was calculated on a weight for weight basis relative to the kits oxazepam calibrators. Fifty-five unrelated drugs were also tested for cross-reaction at a concentration of 100 ug/ml Method Comparison: Urine RIA The DPC Urine Benzodiazepine RIA was compared to a qualitative RIA for benzodiazepines (Roche Abuscreen). The qualitative procedure uses 100 ng/ml oxazepam as a positive/ negative cut-off. Eighty urine samples were analysed for the presence of benzodiazepines by the both RIA procedures using a 100 ng/ml oxazepam cut-off with both the Roche and DPC kits. Forty-one samples were from patients known to be taking benzodiazepines; 39 samples were from drug-free volunteers. Low Dose Benzodiazepines in Urine Urine samples were collected from patients taking a single dose of flunitrazepam (1 mg; N=5) or lorazepam (1 mg; N=l). Urine was collected for 24 hours after the dose and samples were analysed with the DPC Urine Benzodiazepine RIA using the quantitative method. Paired Plasma and Haemolysed Whole Blood Twenty-seven blood samples were collected into lithium heparin tubes from 894
patients receiving various benzodiazepines (on a regular basis) as part of multi-drug therapy for the relief of chronic pain. Blood samples were also collected from 26 patients or healthy volunteers who declared they were not taking benzodiazepines. Samples were split into two: one aliquot was centrifuged and plasma collected; the other was retained as whole blood and subjected to three cycles of freeze/thawing to cause haemolysis of red cells. Plasma and haemolysed whole blood samples were then analysed using the DPC Double Antibody Serum Benzodiazepines RIA. RESULTS Specificity The RIA has a wide class-specific cross-reactivity for the benzodiazepines (Tables 1 & 2). Fifty-five other non-related compounds were not detectable even at 100 ug/ml. TABLE 1 Specificity: Urine Benzodiazepine RIA TABLE 2 Specificity: Serum Benzodiazepine RIA Method Comparison: Urine RIA There was 100% agreement between the two methods in classifying the 80 urine samples as positive or negative relative to the 100 ng/ml oxazepam cut-off. Both kits correctly identified the 39 drug-free volunteers as negative and the 41 samples from patients taking benzodiazepines as positive. Low Dose Benzodiazepines Samples gave detectable amounts of benzodiazepines (ie above the lowest calibrator: greater than 10 ng/ml oxazepam equivalents) for up to 12 hours in each of the six patients after the administration of either 1 mg lorazepam (N=l) or 1 mg flunitrazepam (N=5). Patient 1 taking flunitrazepam had a concentration of 34 ng/ml oxazepam equivalents at 14 hours post dose; similar figures for the other fours patients taking flunitrazepam were: 56 ng/ml at 13 hours; 13 ng/ml at 14 hours; 31 ng/ml at 14.5 hours and 58 ng/ml at 12 hours. The single patient taking lorazepam had a urinary benzodiazepine concentration of 25 ng/ml at 13 hours post dose. Paired Whole Blood & Plasma All 26 paired blood and plasma samples from volunteers and patients not taking benzodiazepines gave undetectable levels of benzodiazepines. The RIA counts were similar for these samples to the counts of the zero calibrator which demonstrates a lack of matrix effect on the assay. Benzodiazepines were detectable in 26 of the plasma samples and 25 whole blood samples from the 27 patients taking benzodiazepines (Table 3). 895
TART.F. 3 Paired Plasma & Whole Blood Patient Benzo Dose Time Plasma Blood Ratio ID -diazepines (mg) since (ng/ml)(ng/ml) P/B last dose (hr:min) 1 Diazepam 10 59:10 430 136 3.2 2 Temazepam 10 14:25 1168 500 2.3 3 Temazepam 20 13:55 1956 988 2.0 5 Temazepam 10 13:20 1048 836 1.3 6 Temazepam 10 19:00 289 168 1.7 7 Temazepam 20 11:00 96 71 1.4 *8 Tem/Diaz 10 >100 42 14 3.0 10 Nitrazepam 15 11:30 3578 1491 2.4 13 Temazepam 20 18:00 2028 1169 1.7 14 Temazepam 60 13:45 6544 2538 2.6 18 Triazolam 0.06 18:00 <10 <10-20 Nitrazepam 10 11:45 120 49 2.4 24 Nitrazepam 5 >100 13 <10-25 Diazepam 10 >100 56 28 2.0 29 Temazepam 10 18:30 and Diazepam 2 4:30 1306 682 *31 Tem/Diaz 10 >100 19 15 1.3 34 Temazepam 60 14:30 47500 10360 4.6 37 Temazepam 40 18:15 2058 720 2.9 38 Diazepam 20 13:15 1100 527 2.1 39 Nitrazepam 10 13:40 112 98 1.4 40 Diazepam 10 13:50 812 450 1.8 41 Temazepam 10 12:30 869 330 2.6 47 Diazepam 10 13:15 1026 672 1.5 50 Temazepam 20 12:10 2154 804 2.7 54 Temazepam 20 9:45 2552 1242 2.1 56 Diazepam 5 12:10 299 149 2.0 75 Diazepam 10 12:00 1010 560 1.8 * patients 8 and 31 were in patients prescribed benzodiazepines for use "as required" but none had been taken for greater than 100 hours. The average plasma/blood ratios for samples from patients taking a single of benzodiazepine were: for diazepam (5, 10 or 20 mg) 2.1, n=7; for temazepam (10, 20, 40 or 60 mg) 2.3, n=12; and for nitrazepam (5, 10 or 15 mg) 2.0, n=3. Table 3 gives actual values for the 27 paired samples from patients taking benzodiazepines. DISCUSSION Both kits (serum and urine) were broadly cross-reactive within the benzodiazepine class and were able to satisfactorily detect the presence of various benzodiazepines in urine, plasma or haemolysed whole blood. 896
The ratio of benzodiazepine concentrations in plasma to whole blood was generally of the order of 2:1. One paired sample of blood and plasma and one separate blood sample in the paired whole blood/plasma study had undetectable levels of benzodiazepines. The paired sample was from a patient who had taken only 0.06 mg triazolam (1/2 tablet) 18 hours previously and this together with the low cross-reactivity of the antibody to triazolam gave an undetectable result. The other negative whole blood had a paired plasma sample containing 13 ng/ml oxazepam equivalents and was from a patient who had taken 5 mg nitrazepam more than 100 hours before the sample was taken. The urine kit was able to classify 80 samples correctly as positive or negative for benzodiazepines and was also able to detect the presence of benzodaizepines in urine for up to 12 hours after a low therapeutic dose of lorazepam or flunitrazepam. 897