Anal. Chem. 1997, 69, 5186-5192
Quantitative Analysis of Despropionyl-Bezitramide, the Active Metabolite of Bezitramide (Burgodin), in Biological Samples by High-Performance Liquid Chromatography with Fluorescence Detection Siegrid M. De Baere, Willy E. Lambert, and Andre´ P. De Leenheer*
Laboratorium voor Toxicologie, Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium
A sensitive high-performance liquid chromatographic procedure with fluorescence detection was developed for the quantitative determination of despropionyl-bezitramide, the active metabolite of bezitramide (Burgodin), in biological samples. Chromatographic separation was achieved on a Hypersil ODS (C18) 5-µm column, using a 31:69 (v/v) mixture of 0.1 M ammonium acetate and methanol/acetonitrile (50:50, v/v)-0.1 M ammonium acetate as the eluent. Internal standardization with Nmethyldespropionyl-bezitramide was used in order to enhance the precision and the accuracy of the method. For the isolation of the compound from biological samples, a liquid-liquid extraction with n-hexane-isoamyl alcohol (93:7 v/v) was performed. Calibration graphs were prepared for blood and urine, and linearity was achieved over a concentration range 1-50 ng/mL. The quantitation limit for despropionyl-bezitramide in blood and urine was 1 ng/mL. At a 10 ng/mL concentration in blood, coefficients of variation of 3.3 and 6.5% were obtained for within-day and between-day precisions, respectively. For urine, the respective coefficient of variation values of 4.3 and 4.9% were obtained. The selectivity and the accuracy of the method were satisfactory. Samples (blood, urine, stomach contents, bile, liver, kidney) from five fatalities that were due to the combined intake of several drugs, including bezitramide, were analyzed and the results are reported. In addition, one blood sample and 14 urine samples from persons suspected of using bezitramide were analyzed, revealing despropionyl-bezitramide concentrations in urine ranging from 1.3 to 72.3 ng/mL. Bezitramide (Burgodin) is a potent, long-acting, orally active narcotic analgesic. The product was introduced and clinically tested around 1970, and it was used for the treatment of severe, chronic pain (e.g., postoperative pain, cancer pain).1 However, nausea, confusion, and side effects of a psychic nature were reported.2 Today, the clinical application of the drug has seriously decreased, but as it is still sold by prescription in Western Europe, it is often abused by drug addicts for its euphoric side effect. In Belgium, even a new increase of the annual sale of bezitramide (1) Meijer, D. K. F.; Hovinga, G.; Versluis, A.; Bro ¨ring, J.; Van Aken, K.; Moolenaar, F.; Wesseling, H. Eur. J. Clin. Pharmacol. 1984, 27, 615-618. (2) Van Der Linden, C.; Hamersma-Van Der Linden, E.; Van Dam, F.; Engelsman, E. J. Drug Res. 1979, 1, 344-351.
5186 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
was observed during the last seven years, following a remarkable decline of the bezitramide sales in the period from 1983 to 1989. After oral administration, bezitramide is slowly absorbed (absorption lag time, 0.5-1.0 h). It is considered a prodrug: a rapid hydrolysis of the propionamide link in the molecule by hydrolytic enzymes in the gastrointestinal tract leads to its main metabolite, despropionyl-bezitramide, which has analgesic properties similar to the parent compound (Figure 1). Maximal plasma concentrations of 4.2-8.2 ng/mL are reached 3.0-3.5 h after administration of a single oral dose of 5 mg of bezitramide. Biliary excretion is the main elimination pathway. Excretion of bezitramide and its main metabolite, despropionyl-bezitramide, in urine is low (0.1-0.3% of the administered dose over 48 h after administration). In urine, however, mainly inactive bezitramide metabolites such as the acidic and basic metabolites (Figure 1) are found.1 Methods for the quantitative determination of the acidic and basic metabolite of bezitramide in urine, have been reported.3-5 Recently, we developed a more sensitive and selective gas chromatographic procedure with nitrogen-phosphorus detection for the basic metabolite.6 By analyzing both the acidic and the basic metabolites of bezitramide in urine, the abuse of the drugsi.e., every use of the drug, not for its clinical effects, but solely for its euphoric side effectscan be assessed without any doubt. However, it is difficult to establish a correlation between the concentration of these inactive metabolites in urine and the overall bezitramide intake, which is very important in the case of fatalities due to drug overdoses. In such cases, the determination of the bezitramide/despropionyl-bezitramide level in blood is of much more significance. A radioimmunoassay has been developed for the determination of the parent compound, bezitramide, and its active metabolite, despropionyl-bezitramide, in biological fluids or tissues. The method is sensitive enough to establish the low bezitramide/ despropionyl-bezitramide levels in the different biological matrixes. Other metabolites of bezitramide are not determined. However, a number of compounds show a substantial cross-reactivity in this assay (e.g., piritramide, diphenoxylate, diphenoxin, a bis(thiophene) analogue, and the compounds with a halogen substituent in the (3) Van Rooy, H.; Kok, M.; Modderman, E.; Soe Agnie, C. J. Chromatogr. 1978, 148, 447-452. (4) De Baere, S.; Lambert, W.; Van Bocxlaer, J.; De Leenheer, A. J. Anal. Toxicol. 1996, 20, 159-164. (5) Van Rooy, H.; Soe Agnie, C. J. Chromatogr. 1978, 156, 189-195. (6) De Baere, S.; Lambert, W.; De Leenheer, A. J. Anal. Toxicol., in press. S0003-2700(97)00618-5 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Metabolism of bezitramide (A) showing despropionyl-bezitramide(B), the acidic metabolite (C), and the basic metabolite (D).
5- or 6-position of the benzimidazole moiety).7 Access to the detailed information on this assay is difficult to obtain, as it is only presented in an internal report and not in a scientific journal. In addition, today, the antibody for this radioimmunoassay is no longer available. From the above remarks it is clear that there is a need for a new method, which is readily accessible and easy to perform, for the determination of bezitramide in blood. Moreover, it will be advantageous if the method can be extended to the analysis of other biological samples. For toxicological purposes, i.e., screening for bezitramide abuse not only in fatalities due to overdoses but also in cases of chronic abuse of the compound, the method has to be sensitive and selective enough to determine the anticipated low bezitramide/despropionyl-bezitramide levels. In this paper, we propose such a method for the determination of despropionyl-bezitramide in different biological matrixes. To enhance the precision and the accuracy of the method, internal standardization was used. A liquid-liquid back extraction with n-hexane-isoamyl alcohol was performed to isolate the analyte from the biological matrix. Finally, a sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) analysis with fluorescence detection was used for the quantitative determination of both despropionyl-bezitramide and the internal standard. EXPERIMENTAL SECTION Apparatus. The HPLC system was composed of a Pye Unicam isocratic LC3-XP pump (Cambridge, U.K.) equipped with an injection valve with a 100-µL sample loop. Chromatographic separation was achieved on a 150 × 4.6 mm i.d. Hypersil ODS (C18) 5-µm column (Alltech Europe, Laarne, Belgium). The elution solvent consisted of a 31:69 (v/v) mixture of solvent A and solvent B. The mobile-phase flow rate was 1 mL/min. The HPLC unit was coupled to a Perkin Elmer LS-4 fluorescence spectrometer with a xenon lamp (Buckinghamshire, U.K.). The excitation and emission wavelengths were fixed at 280 and 310 nm, respectively. The detector was linked to a Spectra-Physics SP4270 reporting integrator (Darmstadt, Germany). (7) Hendriks, R.; Michiels, M.; Heykants, J. Preclinical research report no. R4845/3. Janssen Pharmaceutica, Beerse, Belgium, 1977.
The excitation and emission spectra of despropionyl-bezitramide, its methyl derivative, and pimozide were acquired on a Shimadzu RF-5001PC fluorescence spectrometer (Shimadzu Benelux, Antwerpen, Belgium). Reagents and Standards. All solvents used for the mobile phase (water, methanol, and acetonitrile) were of HPLC grade and were purchased from Prosan (Gent, Belgium). All products (ammonium acetate, sodium hydroxide), reagents (sulfuric acid, concentrated ammonia), and solvents used for the extraction procedure (n-hexane and isoamyl alcohol) were analytical grade and were obtained from UCB (Leuven, Belgium). The ethereal diazomethane solution was synthesized in our laboratory. Elution solvent A consisted of a 0.1 M solution of ammonium acetate in water. Elution solvent B was prepared by adding to a mixture of methanol-acetonitrile (50:50, v/v) a 10 M aqueous solution of ammonium acetate in a 99:1 (v/v) ratio. After appropriate mixing of both elution solvents (31% of A, 69% of B), the resulting mobile phase was filtered through a Nylon 66 (0.2µm pore size) filter and degassed. The standard of despropionyl-bezitramide was a gift from Janssen Pharmaceutica (Beerse, Belgium). A stock solution of 0.1 mg/mL was prepared in methanol. Dilution of the stock solution with methanol yielded the working solutions at concentrations of 10, 1, and 0.1 µg/mL. The internal standard was obtained by preparing the methyl derivative of despropionyl-bezitramide (Figure 2A). After evaporation of 100 µL of the 10 µg/mL working solution, the residue (1 µg of despropionyl-bezitramide) was derivatized overnight at room temperature with 300 µL of an ethereal diazomethane solution. The excess diazomethane was evaporated, and the residue was dissolved in 1 mL of methanol. This experiment was repeated 10 times. An aliquot of each individual internal standard solution was injected onto the HPLC to check for the complete derivatization of despropionyl-bezitramide. This was an absolute criterion for acceptance of Nmethyldespropionyl-bezitramide as the internal standard. By combining the examined internal standard solutions, 10 mL of a 1 µg/mL solution of N-methyldespropionyl-bezitramide was obtained. All stock and working solutions were stored in the refrigerator (6 °C) and were stable for at least six months. Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Figure 2. Chemical structures of two candidate internal standads: (A) N-methyldespropionyl-bezitramide and (B) pimozide.
Samples. The samples (blood, urine, stomach contents, bile, liver, kidney) were from five fatalities that were due to the combined intake of several drugs, including bezitramide. One blood sample was from a patient who was under treatment in a drug rehabilitation clinic. The urine samples were mainly obtained from a drug addiction center (n ) 9). They were from volunteers who were known drug abusers. A few samples (n ) 3) were the result of law enforcement operations against suspected drug abusers. Two samples were from children who were hospitalized because of a suspected bezitramide intoxication. The samples were frozen at -20 °C immediately after receipt. They were thawed just prior to extraction and analysis. Isolation of Despropionyl-bezitramide and the Internal Standard from the Biological Matrix. Blood. To 1 mL of blood were added 15 µL of the internal standard solution, 1 mL of 1 M NaOH, and 3 mL of water. After the sample was vortex mixed for 30 s, it was extracted for 10 min with 6 mL of n-hexaneisoamyl alcohol (93:7, v/v), followed by a 15-min centrifugation step. The organic layer was transferred to another extraction tube containing 2 mL of 0.05 M sulfuric acid. After extraction (10 min) and centrifugation (5 min), the organic phase was rejected. The aqueous layer was alkalinized with 200 µL of concentrated ammonia and again extracted for 10 min with 4 mL of the organic solvent mixture. Finally, the organic layer was transferred to a glass test tube and evaporated to dryness at 40 °C under a gentle stream of nitrogen. The residue was redissolved in 200 µL of a 50:50 (v/v) mixture of the elution solvents A and B, and 100 µL was introduced onto the HPLC. Samples for the calibration curve were treated in a similar way. The calibration samples were prepared by spiking blank blood from healthy individuals not taking medication with despropionyl-bezitramide. The addition of 10 and 50 µL of the 0.1 µg/mL standard working solution and of 10, 25, and 50 µL of the 1 µg/mL standard working solution, resulted in despropionyl-bezitramide concentrations of 1, 5, 10, 25, and 50 ng/mL, respectively. Urine. To 1 mL of urine were added 15 µL of the internal standard solution and 1 mL of 1 M NaOH. After the sample was vortex mixed, it was analyzed after following the same extraction procedure as for blood. Samples for calibration were treated in a 5188
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
similar way. They were prepared by spiking blank urine from healthy individuals not taking medication with the same despropionyl-bezitramide concentrations as previously mentioned for blood. Other matrixes. To 1 mL of stomach contents or bile or 1 g of tissue homogenate, all diluted appropriately with water, was added 15 µL of the internal standard solution. For the analysis of the stomach contents and bile, the extraction procedure for urine was followed. The tissue homogenates were analyzed according to the extraction procedure for blood. Quantitative results for the analysis of stomach contents and bile and for tissue homogenates were obtained using the calibration graphs for urine and blood, respectively. Method Evaluation. The linearity of the method was evaluated using the calibration samples, as previously described. Peak area ratios between despropionyl-bezitramide and the internal standard were plotted against the corresponding despropionylbezitramide concentration. Weighted linear regression was used to calculate the calibration curve. Precision was evaluated by analyzing aliquots from a blank blood and urine pool spiked with despropionyl-bezitramide at three different concentrations (1, 10, and 50 ng/mL). The samples were analyzed on the same day (n ) 6, within-day precision) and over 6 consecutive days (between-day precision). For the determination of the extraction recovery, blank blood and urine samples spiked with despropionyl-bezitramide at the same concentrations as used for the precision evaluation experiment were analyzed according to the procedures described above. Three replicate samples were analyzed for each concentration level. The internal standard, however, was added to the sample after extraction, but prior to injection onto the HPLC. The extraction recovery was then calculated for each concentration level with respect to a corresponding standard mixture (despropionyl-bezitramide-internal standard) that was not extracted but directly injected onto the HPLC. The limit of quantification (LOQ) was determined by analyzing blank blood and urine samples enriched with despropionylbezitramide at concentrations of 2, 1 and 0.5 ng/mL. The LOQ was defined as the lowest concentration of despropionyl-bezitramide that could be quantitated with an error of less than 20% and that could be recognized by the detector with a signal-to-noise ratio of 10.8 The LOQ was also established as the lowest point of the calibration graph. In order to investigate the selectivity of the analysis, several drugs were injected onto the HPLC at a concentration of 1.0 µg/ mL (0.5 µg on column). Solutions of these drugs were prepared in methanol. After solvent evaporation and redissolution of the residue in a 50:50 (v/v) mixture of eluents A and B, each drug was chromatographed under the same conditions as despropionylbezitramide. When retention behavior resulted in possible interference, for either despropionyl-bezitramide or the internal standard, the compound was added to water, extracted, and rechromatographed. The overall accuracy of the method was evaluated by analyzing control samples and relating their quantitative results to the spiked concentrations. These control samples were prepared by spiking blank blood and urine with despropionyl-bezitramide at concentra(8) Dell, D. Analysis for Drugs and Metabolites, including Anti-infective Agents; Guildford Academic Associates: Guildford, U.K., 1990; pp 9-22.
tions of 15 and 30 ng/mL, using volumetric material different from that employed for the preparation of the calibration samples. Safety Considerations. General guidelines for work with organic solvents, acids, and bases were respected. All operations with diazomethane (preparation of the reagent, methylation of despropionyl-bezitramide) were performed under a fume hood and in a well-ventilated room. Blood and urine and other samples of human origin were handled as potentially infectious. RESULTS AND DISCUSSION In order to enhance the precision and the accuracy of the analysis, internal standardization was preferred. Pimozide and N-methyldespropionyl-bezitramide, two structural analogues of despropionyl-bezitramide (Figure 2A,B), were evaluated as candidate internal standards. As the excitation and emission spectra of both molecules were very similar to those of despropionylbezitramide, the detector signal obtained at excitation and emission wavelengths of 280 and 310 nm, respectively, was of comparable intensity for all three compounds. However, when present in high concentrations, despropionyl-bezitramide did not elute baseline separated from pimozide. In addition, pimozide was a drug that could be part of a patient’s therapeutic regimen and therefore could be present in the original samples. N-Methyldespropionyl-bezitramide eluted clearly separated from the active metabolite of bezitramide and was no drug or a metabolite of bezitramide that could be present in the original samples. By performing the methylation of despropionyl-bezitramide on small amounts of the compound and in multiple steps, the derivatization reaction was allowed to proceed quantitatively. Hence, the absolute criterion for acceptance of N-methyldespropionyl-bezitramide as the final internal standard was met. The quality of the internal standard was checked at the beginning of each analysis day by injection onto the chromatographic system (Figure 3). No deterioration of the internal standard was observed during at least six months. Figure 4 depicts the chromatograms of the HPLC analysis of blank blood, blank urine, and blood and urine from a fatality that was due to the combined intake of several drugs (opiates, barbiturates, benzodiazepines, and tricyclic antidepressants), together with bezitramide. As can be seen, despropionyl-bezitramide and the internal standard eluted within 10 min and as clearly separated and symmetrical peaks. Hence, N-methyldespropionyl-bezitramide, chosen as the internal standard for its similar structure and consequently analogous physicochemical properties, obviously met all the requirements for an appropriate internal standard: it eluted closely to the analyte, behaved similarly during the sample preparation procedure and was detectable under the same conditions as the analyte. In addition, it was absent in the original samples and it proved stable during sample manipulations.9 The proper choice of the mobile-phase elution strength and composition was confirmed by the short elution time and the symmetrical peak shape obtained for both despropionyl-bezitramide and the internal standard. For the determination and quantitation of the low levels of despropionyl-bezitramide in biological samples, fluorescence detection was selected. Due to their strong natural fluorescent properties, despropionyl-bezitramide and the internal standard could easily be detected by this technique without any preceding derivatization step. Moreover, fluorescence detection is much (9) Mehta A. J. Clin. Pharm. Ther. 1989, 14, 465-473.
Figure 3. Chromatogram of the N-methyl derivative of a standard despropionyl-bezitramide (IS). The absence of a peak at the eluting position of despropionyl-bezitramide (DB) confirmed that the derivatization was complete.
more sensitive and selective than the classical UV detection technique. For our quantitative purposes, however, the linear dynamic range of the detector had to be established. This was determined by analyzing increasing amounts of despropionylbezitramide and by plotting the resulting peak area against the amount injected. A linear response was obtained over a range from 0.5 to 25 ng of the compound on the HPLC column, corresponding with a concentration range from 1 to 50 ng/mL. The identification of a peak in the chromatogram of an extract as despropionyl-bezitramide was performed by comparing the capacity factor of the peak with that of a despropionyl-bezitramide standard. Capacity factors for despropionyl-bezitramide and the internal standard were fairly constant during the course of many analyses. In an early stage of our sample preparation development experiments, each sample was hydrolyzed in alkaline medium prior to extraction, in order to completely convert bezitramide to its active metabolite despropionyl-bezitramide. Hence, the quantitative result obtained by analysis of despropionyl-bezitramide in biological samples was a measure of the total bezitramide/ despropionyl-bezitramide level and of the total amount of active drug in that matrix. During the course of the experiments, however, it became clear that such a hydrolysis step was superfluous. As the extraction procedure was performed at a pH that was alternately strongly alkaline and strongly acidic, rapid hydrolysis of the propionyl group of bezitramide occurred during the sample preparation procedure, resulting in a complete conversion of the parent compound to despropionyl-bezitramide. The hydrolysis of urine samples was not necessary to release possibly conjugated despropionyl-bezitramide, since conjugation of the compound does not occur.5 Because the concentrations of Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Figure 4. HPLC analysis of (A) blank blood and (B) blank urine, showing the eluting position of despropionyl-bezitramide (DB) and the internal standard (IS), (C) blood, and (D) urine from a fatality, showing DB at 14.0 and 3.0 ng/mL, respectively, and the IS.
despropionyl-bezitramide in blood and urine are generally low, an extraction procedure that allowed the isolation of the analytes from the biological matrix with a high recovery and an acceptable selectivity was needed. Therefore, several extraction solvents of different polarities were explored. The best results were obtained with mixtures of n-hexane-isoamyl alcohol in a ratio of 97:393:7 (v/v). Finally, a mixture of n-hexane-isoamyl alcohol in a ratio of 93:7 (v/v) was chosen as the extraction solvent, because a good total extraction recovery (mean value of 86.0% for blood and urine) was obtained. As the concentrations of despropionylbezitramide in biological samples, other than blood and urine, exceeded the linear dynamic range of the detector, appropriate dilution of each matrix with water was necessary. The dilution factor was not constant for a specific matrix but differed from case to case. Therefore, no separate calibration graphs were prepared for the quantitative determination of despropionyl-bezitramide in stomach contents, bile, liver, and kidney, but the curves for blood and urine were used. The calibration curves of despropionyl-bezitramide were constructed using weighted linear regression analysis in order to account for data heteroscedasticy. The curves for blood and urine were linear over the specified range of 1-50 ng/mL and respective correlation coefficients of 0.9962 and 0.9939, or higher, were obtained for the relationship between the peak area ratio of despropionyl-bezitramide and the internal standard and the various calibration concentrations. The y-intercept for each calibration curve was virtually zero, and the coefficients of variation (CV %) for the regression slopes (a) were low (Blood: a ) 0.0850, CV ) 2.5%, n ) 6. Urine: a ) 0.0963, CV ) 1.6%, n ) 6). These linearity data indicate a good day-to-day match for the various calibration curves. The results of the precision evaluation were rather similar for blood and urine. At a 10 ng/mL concentration in blood, coefficients of variation of 3.3 and 6.5% were obtained for within-day repeatability and between-day reproducibility, respectively. For urine, respective CV values of 4.3 and 4.9% were obtained. For 5190
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
Table 1. HPLC Retention Data of Drugs Evaluated as Possible Interferents drug
k′
caffeine 6-monoacetylmorphine benzoylecgonine acetylcodeine heroine amphetamine 3,4-methylenedioxyethylamphetamine dextromoramide hydrocodone pentazocine viloxazine citalopram ibuprofen methaqualone fluoxetine desipramine maprotiline imipramine butriptyline trimipramine nortriptyline despropionyl-bezitramide mianserine lofepramine internal standard bromazepam, nitrazepam, flurazepam, diazepam, oxazepam, lorazepam, medazepam, flunitrazepam, ketazolam, alprazolam, chlordiazepoxide, nordiazepam, dibenzepine, opipramole, melitracene, trazodone, doxepine, fluvoxamine, amitryptiline, sertraline, apomorphine, oxycodone, dihydrocodeine, methadone, fentanyl, d-propoxyphen, pethidine, indomethacin, phenylbutazon, acetaminophen, acetylsalicylic acid, cocaine, meprobamate, nicotine, phenobarbital, secobarbital, hexobarbital
0.05 0.13 0.15 0.31 0.34 0.35 0.40 0.49 0.50 0.53 0.67 0.75 0.76 0.87 1.61 1.65 1.72 2.53 3.27 3.34 3.53 3.65 3.90 4.01
a
5.47 nda
nd, not detected.
the other concentrations tested (1 and 50 ng/mL), CV values were below 11%. This indicated that the reproducibility of the method was acceptable over the studied concentration range.9 The extraction recovery was determined within the concentration range of 1-50 ng/mL. The results obtained (84.2-88.2% for
Table 2. Despropionyl-bezitramide Concentrations in Biological Matrixes from Human Subjects Suspected of Bezitramide Abuse
subject
a
gender
age (years)
1 2 3
a Mc Fc
a 24 19
4
Mc
a
5
Mc
33
6 7 8
M M M
1.5 33 36
9
F
38
10
M
26
11
M
34
12 13 14 15
M F a Mc
35 40 a 42
16 17
F M
1.6 a
18
F
a
other drugs demonstrated
blood (ng/mL)
urine (ng/mL)
stomach contents (µg/mL)
bile (ng/mL)
liver (ng/g)
kidney (ng/g)
benzodiazepines benzodiazepines cannabinoids amphetamines benzodiazepines opiates benzodiazepines cannabinoids opiates barbiturates benzodiazepines tricyclic antidepressants d cocaine opiates cannabinoids opiates benzodiazepines d d opiates cannabinoids cocaine benzodiazepines cannabinoids benzodiazepines cannabinoids cocaine opiates opiates benzodiazepines barbiturates d opiates cocaine benzodiazepines opiates cocaine benzodiazepines
40.8 27.2 7.7
b b 13.4
b 38.1 0.2
b 172.5 1388.5
b 895.5 386.9
b 52.6 73.7
14.4
2.7
b
b
467.4
b
14.0
3.0
5.2
b
185.9
66.9
b b b
19.0 9.8 55.5
b b b
b b b
b b b
b b b
b b b b b
6.4e
b
b
b
b
6.9e 7.1e 2.8
b b b
b b b
b b b
b b b
b
23.3
b
b
b
b
b b b b
11.3 5.7 4.8 72.3
b b b b
b b b b
b b b b
b b b b
b b
2.0 3.3
b b
b b
b b
b b
b
1.3
b
b
b
b
Unknown. b Matrix not available. c Postmortem samples. d Negative. e Same person, different samples over a period of time.
blood and 80.5-90.9% for urine) were independent of the concentration of the particular sample. The LOQ was established at 1 ng/mL for blood and urine, as signal-to-noise ratios of 9.6 and 9.8 were observed, respectively. Moreover, this concentration was quantitatively measurable with reproducibilities of 11.0 and 9.9% for blood and urine, respectively, which were substantially better than the allowed coefficient of variation of 20%.8,9 The method proved to be highly specific for despropionylbezitramide and the internal standard. As shown in panels A and B of Figure 4, there were no interfering peaks from endogenous compounds on the elution position of the internal standard. In the eluting zone of despropionyl-bezitramide, a small endogenous peak was observed. However, the endogenous compound eluted baseline separated from low concentrations (1-10 ng/mL) of despropionyl-bezitramide. Hence, it was possible to clearly distinguish a positive sample from a negative one. As the development of a method suitable for use in a toxicological laboratory was the objective of this study, selectivity considerations were of prime importance. In that respect, several therapeutic and abused drugs, regarded as possible interferences, were analyzed. Table 1 shows the capacity factors of the tested drugs
in ascending order. As can be seen, most of the drugs analyzed were either not retained on the column or determined by the specific fluorescence detector. Some drugs eluted in the first part of the chromatogram (k′
