Journal of Analytical Toxicology, Vol. 16, July/August 1992
Solid-Phase Extractionand HPLC-UV Confirmationof Drugs of Abuse in Urine S.D. Ferrara*, L. T e d e s c h i , G. Frison, and F. C a s t a g n a
Centre of Behavioural and Forensic Toxicology, Istituto Medicina Legale, Via Falloppio 50, University of Padova, Italy
Abstract I A series of six liquid chromatographic methods were developed to confirm the presence of six classes of drugs of abuse In urine. The chromatographic separations were performed with a reversed-phase Ca column, except In the case of morphine, which was separated on a normal phase column, laocratic and gradient elutions, Ion pair technique, and UV detection were employed. Sample pretreatment involved the extensive application of solid-phase extractions and liquid-liquid extractions on solid supports. The specificity and sensitivity enabled the confirmation of morphine, benzoylecgonlne, THC-COOH, amphetamine, and methamphetamlne, six barbiturates, and nine benzodlazeplnes screened positive by EMIT in urine.
equipped with GC/MS instruments because they are costly to acquire and maintain, and the analysts must possess a high level of skill and experience in order to operate the instrument and interpret the data. High pressure liquid chromatography (HPLC) is an alternative chromatographic technique to GC/MS. A number of HPLC methods have been described for the analysis of drugs of abuse in biological fluids, but most relate to individual drugs or groups of structurally related drugs (6--17). The current study proposes a series of reliable HPLC-UV procedures covering the most important drugs of abuse. These are intended for the confirmation of results obtained by EMIT d.a.u, in the analysis of opiates, cocaine, cannabinoids, amphetamines, barbiturates, and benzodiazepines in urine. We also investigated the application of liquid-liquid extractions on a solid support (18) and of solid-phase extractions (19), both on disposable columns, for sample preparation.
Introduction
The detection of drugs of abuse in biological samples, particularly in urine, is one of the primary functions of a clinical or forensic toxicology laboratory (1-3). Particularly in cases where the results are relevant to legal proceedings, confirmatory tests are essential to ensure the accuracy of results. The main requirements of the confirmatory methods are specificity and the capability to separate and identify the drugs of interest and related substances. To ensure reliability and accuracy, the confirmatory tests must be different in chemical and physical principles from those of the screening tests. Immunochemical methods, such as enzyme immunoassay, fluorescence immunoassay, and radioimmunoassay, are generally preferred for screening. Unfortunately, the antibodies used in many immunoassays may cross react with chemically related drugs and metabolites. This is the main reason for the extensive application of chromatographic confirmatory tests. The current tendency is to advocate gas chromatography/mass spectrometry (GC/MS) as the method of choice for confirmation. GC/MS is highly sensitive and specific, and its ability to couple spectral data (using the "full scan" or the selected ion monitoring modes) with retention time data allows a definitive identification of drugs (4,5). Nevertheless, many laboratories are not 9 Authorto whom correspondence shouldbe addressed.
Experimental Materials All solvents used in sample extraction and in mobile phase preparation were analytical and HPLC grade, respectively (E. Merck, Darmstadt, Germany). All reagents used in sample extraction and analysis were analytical grade and were obtained from Merck, with the exception of the enzyme ~-Glucuronidase (No. G-0876), which was purchased from Sigma Chemical Company. The following pure drugs were used as standards: morphine 9 HCI, codeine 9H3PO4, nalorphine ~ HBr, benzoylecgonine ~ 4H20, 11-nor-A9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), amphetamine, methamphetamine, barbital, allobarbital, phenobarbital, butabarbital, pentobarbital, secobarbital (Division of Narcotic Drugs, United Nations, Vienna, Austria), 7-acetamidonitrazepam, 7-aminonitrazepam, 7-aminoflunitrazepam, demoxepam, desmethylchlordiazepoxide, oxazepam, n-hydroxyethylflurazepam, and desmethylflunitrazepam, desmethyldiazepam (Hoffmann-LaRoche, Basel, Switzerland). Solid-phase extraction columns, Adsorbex-Diol 100 mg, and Adsorbex-RP8 100 mg, as well as Extrelut 3 and Extrelut 20 columns, were obtained from Merck.
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Journal of Analytical Toxicology, Vol. 16, July/August 1992
Buffer solutions
Benzoylecgonine. Phosphate buffer (pH 2.1; 0.01 M) for mobile phase was prepared as follows: 0.68 g of sodium dihydrogen phosphate was dissolved in 450 mL of water; the pH was adjusted to 2.1 by addition of conc. phosphoric acid; 0.432 g of dodecyl hydrogen sulfate sodium salt was added and the resulting mixture was diluted with water to obtain a final volume of 500 mL. THC-COOH. Phosphoric acid (0.05M) for mobile phase: 3.4 mL of conc. phosphoric acid was diluted with water to obtain a final volume of 1000 mL. Amphetamines. Phosphate buffer (pH 3; 0.01M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 950 mL of water; 1.3 mL of methanesulfonic acid was added and the pH was adjusted to 3 with 5N potassium hydroxide. The resulting mixture was diluted with water to obtain a final volume of 1000 mL. Barbiturates. Phosphate buffer (pH 5.5; 0.5M): 6.8 g of potassium dihydrogen phosphate was dissolved in 95 mL of water. The resulting solution, after adjusting the pH to 5.5 with dil. phosphoric acid, was diluted with water to obtain a final volume of 100 mL. Phosphate buffer (pH 4.4; 0.01M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 980 mL of water; the pH was adjusted to 4.4 with dil. phosphoric acid and water was added to obtain a final volume of 1000 mL. Benzodiazepines. Acetate buffer (pH 5; 0.2M): The pH of 50 mL of 1M sodium acetate was adjusted to 5 by adding about 14 mL of IM hydrochloric acid and the resulting solution was diluted with water to obtain a final volume of 250 mL. Borate buffer (pH 9.2; 0.1M): 0.618 g of boric acid and 0.746 g of sodium tetraborate were dissolved in 100 mL of water. The pH of 50 mL of this solution was adjusted to 9.2 by addition of 0. IN sodium hydroxide. Phosphate buffer (pH 3.5; 0.01 M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 950 mL of water; 1.3 mL of methanesulfonic acid was added and the pH was adjusted to 3.5 with 5N potassium hydroxide. The resuhing mixture was diluted with water to obtain a final volume of 1000 mL. Preparation of standards Concentrations of the drugs in standard solutions, used for testing the extraction procedures, were prepared by adding appropriate amounts of the stock standards to urine samples from healthy volunteers. Such stock solutions were prepared at concentrations of 10 lag/mL (except nalorphine 100 lag/mL) in methanol. These solutions were stored at 4~ in the dark and were found to be stable for at least six months. Appropriate dilutions of standard solutions, with methanol or with the mobile phases, provided working standards. Urine samples from drug users were obtained from the local hospital or from drug rehabilitation centers.
Extraction procedures Morphine. 0.5 mL of a 100-gg/mL nalorphine 9 HBr methanolic solution (internal standard) and 1 mL of conc. hydrochloric acid were added to 10 mL of urine. The mixture was hydrolyzed for 1 h at 100~ and, after cooling, 0.5 mL of a sat. ammonium sulfate solution was added. The pH was then adjusted to 9 with 25% sodium hydroxide. The solution was diluted with water to obtain a final volume of 20 mL and poured into an Extrelut 20 column. After 10 rain, elution was carried out with 40 mL of a 15% solution of isopropyl alcohol in methylene chloride. The eluate obtained was extracted twice with 3 mL of
218
0.2N hydrochloric acid. The organic phase was discarded and 0.5 mL of the sat. ammonium sulfate solution was added to the aqueous phase and the pH was adjusted to 9.2 with 25% sodium hydroxide. The mixture was again diluted with water to obtain a final volume of 20 mL and treated on a second Extrelut 20 column in the same way as on the first one. The final eluate was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 gL of HPLC mobile phase and a 20-gL aliquot was injected into the chromatograph. Benzoylecgonine. A 3.5-mL aliquot of urine was adjusted to pH 9 with 60 mg of sodium bicarbonate-potassium carbonate (2:1, w/w) and, if necessary, with small amounts of acid or base. The urine specimen was poured into an Extrelut 3 column and, after 10 min, elution was carried out with 15 mL of a 10% solution of isopropyl alcohol in chloroform. The eluate volume was reduced to approximately 100 gI_, with a stream of nitrogen at 40~ and a 5-mL aliquot of methylene chloride was added to the tube. The organic solution obtained was passed through an Adsorbex-Diol 100-mg column previously conditioned with 2 mL of methylene chloride. The column was then washed with 1 mL of methylene chloride and 4 mL of diethyl ether. Benzoylecgonine was eluted with two 500-gL aliquots of methanol. This final organic solution was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 IlL of HPLC mobile phase and a 20-1aL aliquot was injected into the chromatograph. THC-COOH. A 5-mL aliquot of urine was heated for 20 min at 60~ after adding 0.5 mL of 10N potassium hydroxide. The hydrolyzed sample was cooled to room temperature and then extracted with 10 mL of a 10% solution of ethyl acetate in nhexane. The organic phase was discarded and the remaining aqueous phase was acidified (pH 3.5) with 6N hydrochloric acid and subsequently passed through an Adsorbex RP8 100-rag column previously conditioned with 2 mL of methanol and 4 mL of water. The column was then washed with 5 mL of water and two l-mL aliquots of a 20% solution of acetone in water. THCCOOH was eluted with two 1-mL aliquots of a 10c/~' solution of ethyl acetate m n-hexane. The obtained organic solution was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 bt.L of HPLC mobile phase and a 20-btL aliquot was injected into the chromatograph. Amphetamines. The pH of a 3-mL aliquot of urine was adjusted to 11 with 10N potassium hydroxide. The sample was poured into an Extrelut 3 column, one drop 3N hydrochloric acid was poured into the collection tube and, after 10 min, elution was carried out with 15 mL of n-hexane. The eluate was evaporated to dryness under a stream of nitrogen at 35~ 1.5 mL of an 8% aqueous solution of sodium bicarbonate and 1 mL of a 0.5% aqueous solution of sodium naphthoquinone-4-sulfonate (NQS) were added to the residue. The resulting mixture was heated at 70~ for 20 min. After cooling, it was extracted with 5 mL of carbon tetrachloride and the resulting organic phase was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 uL of HPLC mobile phase and a 20-uL aliquot was injected into the chromatograph. Barbiturates. 1 mL of phosphate buffer (pH 5.5; 0.5M) was added to 2 mL of urine. The resulting mixture was poured into an Extrelut 3 column and, after 10 rain, elution was carried out with 15 mL of a 5% solution of isopropyl alcohol in methylene chloride. The eluate was evaporated to dryness under a stream of nitrogen at 40~ and the residue was reconstituted with 100 of HPLC mobile phase. 20 ,u.Lof this final solution was injected into the chromatograph.
Journal of Analytical Toxicology, Vol. 16, July/August 1992
Benzodiazepines. A 2.5-mL aliquot of urine was adjusted to pH 5 by adding dropwise dil. hydrochloric acid. 0.4 mL of acetate buffer (pH 5; 0.2M) and 0.05 mL of 13-glucuronidase were then added. The mixture was incubated for 5 h at 45~ After cooling, the hydrolyzed sample was adjusted to pH 8-9 with 25% sodium hydroxide and subsequently poured into an Extrelut 3 column. After 10 min, elution was carried out with 15 mL of a 10% solution of isopropyl alcohol in chloroform. The eluate was evaporated to dryness under a stream of nitrogen at 40~ and the residue was reconstituted with 2 mL of borate buffer (pH 9.2; 0.1M). The resulting solution was extracted twice with 3 mL of diethyl ether and the organic phase was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 laL of HPLC mobile phase and a 20-laL aliquot was injected into the chromatograph.
Enzyme Immunoassay Urine specimens were screened for the presence of opiates, benzoylecgonine, THC-COOH, amphetamines, barbiturates, and benzodiazepines by EMIT d.a.u, according to the manufacturer's procedures. Samples were processed using a Syva Model 1500 automatic pipetter--diluter connected to a Syva Autocarousel, a Gilford Stasar S-Ill spectrophotometer and a Syva CP-5000 Plus clinical processor. The cutoff levels set for the above assays were 300 ng/mL (except cannabinoids, 100 ng/mL) and corresponded to the concentration of the drugs in Syva's "low calibrator" solutions.
Results a n d D i s c u s s i o n Apparatus and chromatographic conditions The HPLC apparatus consisted of a Merck Hitachi L 6200 intelligent pump, L 4200 UV-VIS detector, D 2000 Chromatointegrator, and a Rheodyne 71-25 injection valve with a 20-~aL sample loop. A Hibar analytical column (Merck), 250 x 4 mm, packed with Lichrospher Si I00 (5 wn) and a Lichrocart 4-4 pre-column (Merck) packed with Lichrosorb (5 ~n) were used for morphine detection. A Hibar analytical column (Merck), 250 x 4 mm, packed with Lichrospher 100 RP8 (5 ~trn) and a Lichrocart 4-4 pre-column (Merck) packed with Lichrospher 100 RP8 (5 larn) were used for benzoylecgonine, THC-COOH, amphetamine, barbiturate, and benzodiazepine detection. The eluents were monitored at 225,233, 211,460, 212, and 234 nm, respectively, and for morphine, benzoylecgonine, THCCOOH, amphetamines, barbiturates, and benzodiazepines. All chromatographic separations were accomplished at room temperature using the following mobile phases. Morphine. n-Hexane-methylene chloride-methanol containing diethylamine (0.75%, v/v) (72.5:20:7.5, v/v/v). Flow rate: 1.35 mL/min. Benzoylecgonine. Phosphate buffer (pH 2.1; 0.01M) containing 0.003M dodecyl hydrogen sulfate sodium salt-acetonitrile (60:40, v/v). Flow rate: 1 mL/min. THC-COOH. 0.05M phosphoric acid-acetonitrile (35:65, v/v). Flow rate: 0.8 mL/min. Amphetamines. Phosphate buffer (pH 3; 0.01M) containing 0.02M methanesulfonic acid-acetoni~le (45:55, v/v). Flow rate: 1 mL/min. Barbiturates. Phosphate buffer (pH 4.4; 0.01M)-acetonitrile with the following gradient composition: t = 0 min, 70:30 v/v; t = 8 min, 60:40 v/v; t = 14 min, 60:40 v/v; t = 15 min, 70:30 v/v. Flow rate: 1 mL/min. Benzodiazepines. Phosphate buffer (pH 3.5; 0.01M) containing 0.02M methanesulfonic acid-acetonitrile, with compositions suitable for two alternative analyses. Benzodiazepine metabolites (Mobile phase 1). t = 0 min, 70:30 v/v; t = 1 min, 70:30 v/v; t = 7 min, 60:40 v/v; t = 16 min, 60:40 v/v; t = 18 min, 70:30 v/v. Flow rate: 1 mL/min. Oxazepam and desmethyldiazepam (Mobile phase 2). 55:45 v/v; Flow rate: 1 mL/min. All liquid chromatographic solvents and buffer solutions were filtered through ANODISC 47 (0.2 ~un) filters (Merck) and degassed under vacuum with stirring before use. Glassware for the THC-COOH detection was silanized before use to prevent adsorption of the drug metabolite on borosilicate glass.
Typical chromatograms obtained by HPLC analysis of the above mentioned individual drugs or classes of drugs are reported in Figures 1-6. Every figure shows the analysis of (a) a standard solution containing a single drug or a mixture of drugs, (b) an extract of a urine sample spiked with the substance(s) under analysis, and (c) an extract of a corresponding EMIT positive urine sample.
a
b
c
LL Figure 1. Liquid chromatograms of (a) a standard mixture of opiates (1-3), (b) an extract of a urine sample spiked with 1-3 (5 lag/mL each), and (c) an extract of an EMIT positive urine sample for opiates (containing 1:5 ~/mL). Peak assignments: 1 = nalorphine, 2 = codeine, and 3 = morphine.
a
b
c
Figure 2. Liquid chromatograms of (a) a standard solution of benzoylecgonine (1), (b) an extract of a urine sample spiked with 1 (1 ~/mL), and (c) an extract of an EMIT positive urine sample for cocaine metabolite.
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Journal of Analytical Toxicology, Vol. 16, July/August 1992
a
b
a ".
c
b I
r'
Figure 3. Liquid chromatograms of (a) a standard solution of 11-nor-tPtetrahydrocannabinol-9-carboxylic acid (1), (b) an extract of a urine sample spiked with 1 (0.4 lag/mL), and (c) an extract of an EMIT positive urine sample for cannabinoids.
;2~3
2
"3
c
!2 3
Figure 5. Liquid chromatograms of (a) a standard mixture of six barbiturates (1-6), (b) an extract of a urine sample spiked with 1-6 (10 lag/mL each), and (c) an extract of an EMIT positive urine sample for barbiturates. Peak assignments: 1 = barbital, 2 = allobarbital, 3 = phenobarbital, 4 = butabarbital, 5 = pentobarbital, and 6 = secobarbital.
a 1
~
"
b
r
d
b
,
e
e 9 I
1112
2
8 9 2 3
__t_T_ Figure 4. Liquid chromatograms of (a) a standard mixture of amphetamines derivatized with NQS, (b) an extract of a urine sample spiked with amphetamine and methamphetamine (1 ~g/mL each) after derivatization with NQS, and (c) an extract of an EMIT positive urine sample for amphetamines (after derivatization with NQS). Peak assignments: 1 = NQS-derivatized amphetamine and 2 = NQS-derivatized methamphetamine.
Extractions
An initial step common to all the proposed procedures consisted of the adjustment of the extraction pH. The best recoveries were obtained at pH 9 for the amphoteric compounds, morphine and benzoylecgonine, and at strong and mildly acidic pH, respectively, for THC-COOH and barbiturates. The chromatographic analyses of morphine, THC-COOH, and benzodiazepines could be efficiently performed only after a prior hydrolytic treatment of the corresponding urine specimens in order to obtain the cleavage of the relative glucuronic conjugates. In this context acidic hydrolysis was used for morphine sample treatment, although enzymatic hydrolysis can be similarly applied. Alkaline hydrolysis proved to be more efficient for THCCOOH assay with respect to cleanliness of the extracts and reaction times, according to previous papers (20-21). The application of enzymatic hydrolysis was essential for benzodiazepinic compounds because of their widely known relative instability in acidic conditions. As previously reported (18), the extensive use of the Extrelut extraction columns as an alternative to the conventional liquid-liquid extractions speeded up sample preparation (enabling more samples to be processed per day),
220
Figure 6. Liquid chromatograms of (a) a standard mixture of nine benzodiazepine metabolites (1-9) (Mobile phase 1), (b) a standard mixture of 6 and 9 (Mobile phase 2), (c) an extract of a urine sample spiked with 1-9 (1 i.tg/mL each) (Mobile phase 1 ), and (d) an extract of an EMIT positive urine sample for benzodiazepines (Mobile phase 2) Peak assignments: 1 = 7-acetamidonitrazepam, 2 = 7-aminonitrazepam, 3 = 7-aminoflunitrazepam, 4 = demoxepam, 5 = desmethylchlordiazepoxide, 6 = oxazepam, 7 = N-hydroxyethylflurazepam, 8 = desmethylflunitrazepam, and 9 = desmethyldiazepam.
eliminated incidental emulsions, and allowed high extraction recoveries to be obtained. In particular, the use of n-hexane as the liquid extraction phase resulted in the efficient extraction of amphetamines and reduced the co-extraction of polar urinary components. Similarly, for the barbiturates, the pH adjustment to 5.5 of the urine specimens avoided the extraction of interfering basic compounds and did not require additional purification steps. Solid-phase extractions were efficiently employed for the isolation of benzoylecgonine and THC-COOH. In the former case, the hydrogen bondings and dipole-dipole interactions between the analyte molecules and the diol groups on the sorbent (Adsorbex-Diol column) allowed the polar cocaine metabolite present in a nonpolar solvent of dissolution (CH2C12) to be selectively retained. In the latter case, the THC-COOH was selectively retained by the solid nonpolar sorbent (Adsorbex RP8 column) by means of hydrophobic interactions between the octyl chains of the stationary phase and the alkyl and aromatic moieties of the analyte molecules. The selectivity and specificity, important pe-
Journal of AnalyticalToxicology,Vol. 16, July/August1992
culiarities of the solid-phase extraction, were exploited with the optimization of the column washing steps: diethyl ether, on the one hand, as well as water and a 20% solution of acetone in water, on the other, removed some sample matrix interferences (salts, ionic compounds, UV-absorbing urine components, etc.), enabling relatively clean final extracts to be obtained containing the substance(s) under analysis. With respect to the proposed HPLC analyses, ultraviolet detection proved to be effective. However, a derivatization reaction was necessary for the analysis of urine samples containing amphetamines because these amines show only relatively weak absorption bands in UV range. The reaction with NQS formed highly colored derivatives of amphetamine and methamphetamine that showed strong absorption bands in UV (248-280 nm) and visible ranges (450-460 nm). HPLC analysis Reversed-phase liquid chromatography with a C 8 column was the preferred technique for the analysis of the drugs under consideration. A normal phase column was used only for morphine. In this context the capability of detecting the codeine proved to be particularly useful because of the high cross-reactivity between morphine, codeine, and their glucuronides in immunoassays. The ion-pair HPLC technique obtained the best chromatographic performances: the benzoylecgonine, the amphetamine derivatives, and the benzodiazepinic compounds, all positively charged at the acidic pH of the relative mobile phases, form neutral ion pairs with dodecyl hydrogen sulfate sodium salt or methanesulfonic acid, permitting efficient hydrophobic interactions between the analytes and the stationary phase. Mobile phases with solvent composition gradients generated fairly good chromatographic separations in barbiturate and benzodiazepine analyses. With regard to the benzodiazepine analysis, two mobile phases with different compositions were found to be useful and were Table I. Comparison between GC/MS and H P L C - U V as Methods of Confirmation for EMIT Positive Results EMIT d.a.u. assay Opiates
Positive Correlation(%) samples GC/MS HPLC-UV
Notes
125
96
92
70
98.5
97.1
102 54
98 98.2
89.2 98.1
Barbiturates
32
100
100
All samplespositive for phenobarbital
Benzodiazepines
80
92.5
87.5
2 samplescontaining benzodiazepinesnot included in the HPLC assay
Cocaine metabolite Cannabinoids Amphetamines
3 samplespositive for codeine by both confirmation techniques
4 samplespositive for methamphetamine by both confirmation techniques
consequently adopted for a general screening of various benzodiazepine metabolites and for oxazepam and desmethyldiazepam detection. The HPLC methods were developed merely to confirm the presence of drugs of abuse in urine and not to quantify them. Consequently, the use of internal standards was not considered strictly necessary. However, because the positive identification of the substances under analysis is based on retention times, a routine check of their relative daily variability is suggested. This can be accomplished by simply injecting the pure standards at regular intervals. Validation of detections The liquid chromatographic procedures showed recoveries of the substances in the range 75-85% (n -- 5, CVs < 12%). Specificity and sensitivity were optimized to allow the detection of urinary levels of the substances under analysis near the corresponding cutoff levels of the EMIT d.a.u, assays used as screening tests. The limits of detection (K -- 3) of the above mentioned HPLC procedures were as follows: 100 ng/mL morphine; 75 ng/mL benzoylecgonine; 50 ng/mL THC-COOH; 60 ng/mL amphetamine and methamphetamine, 100 ng/mL phenobarbital and 150 ng/mL secobarbital; and 100 and 200 ng/mL of oxazepam, using mobile phases 2 and 1 respectively. A consistent number of EMIT positive urine samples for the six classes of drugs of abuse were analyzed for confirmation purposes by HPLC and GC/MS procedures routinely employed in our laboratory (5). These duplicate analyses were made to evaluate the applicability of the liquid chromatographic procedures as effective analytical tools in confirmatory tests. The results (see Table I) demonstrate that the proposed HPLC procedures can offer performances comparable to those typically obtainable by the majority of GC/MS procedures. The following is a list of drugs found to be present by GC/MS in association with the reported drugs of abuse in more than 300 samples analyzed for the correlation studies. Under the chromatographic conditions applied, no interferring peaks were observed.
Drugs Found Not to Interfere with the HPLC Procedures Acetaminophen Acetylsalicylicacid Amitriptyline Buprenorphine Caffeine Carbamazepine Chlorpromazine Desipramine Dextromethorphan Doxepin Ephedrine Fenfluramine Imipramine Lidocaine Loxapine Meperidine Methadone Methaqualone Naloxone Naltrexone Nicotine
Orphenadrine Oxicodone Papaverine Pentazocine Phendimetrazine Phenmetrazine Phentermine Phenylpropanolamine Phenytoin Primidone Procaine Prometazine Propoxyphene Propyphenazone Theobromine Theophylline Trazodone Triflupromazine Trimethoprim Trimipramine
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Conclusions
The reported procedures prove the general applicability of liquid chromatography with UV detection for confirmation of drugs of abuse in urine. Especially when the sample treatment is developed to reduce the presence of interfering UV-absorbing compounds and adequate chromatographic conditions are chosen, liquid chromatography with UV detection is a valid analytical tool which merits serious consideration for specific and sensitive identifications in routine cases. Furthermore, the analytical system consisting of immunoassay screening and HPLC confirmation tests can be considered sufficiently reliable and specific to be applied in clinical and forensic toxicology laboratories for detection of drugs of abuse in urine. Nonetheless, GC/MS actually remains the ideal analytical means of confirmation of the presence of drugs in biological fluids, exhibiting generally better confirmatory power than that of other techniques, such as HPLC.
9.
10.
11.
12.
13.
14.
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Solid-Phase Extractionand HPLC-UV Confirmationof Drugs of Abuse in Urine S.D. Ferrara*, L. T e d e s c h i , G. Frison, and F. C a s t a g n a
Centre of Behavioural and Forensic Toxicology, Istituto Medicina Legale, Via Falloppio 50, University of Padova, Italy
Abstract I A series of six liquid chromatographic methods were developed to confirm the presence of six classes of drugs of abuse In urine. The chromatographic separations were performed with a reversed-phase Ca column, except In the case of morphine, which was separated on a normal phase column, laocratic and gradient elutions, Ion pair technique, and UV detection were employed. Sample pretreatment involved the extensive application of solid-phase extractions and liquid-liquid extractions on solid supports. The specificity and sensitivity enabled the confirmation of morphine, benzoylecgonlne, THC-COOH, amphetamine, and methamphetamlne, six barbiturates, and nine benzodlazeplnes screened positive by EMIT in urine.
equipped with GC/MS instruments because they are costly to acquire and maintain, and the analysts must possess a high level of skill and experience in order to operate the instrument and interpret the data. High pressure liquid chromatography (HPLC) is an alternative chromatographic technique to GC/MS. A number of HPLC methods have been described for the analysis of drugs of abuse in biological fluids, but most relate to individual drugs or groups of structurally related drugs (6--17). The current study proposes a series of reliable HPLC-UV procedures covering the most important drugs of abuse. These are intended for the confirmation of results obtained by EMIT d.a.u, in the analysis of opiates, cocaine, cannabinoids, amphetamines, barbiturates, and benzodiazepines in urine. We also investigated the application of liquid-liquid extractions on a solid support (18) and of solid-phase extractions (19), both on disposable columns, for sample preparation.
Introduction
The detection of drugs of abuse in biological samples, particularly in urine, is one of the primary functions of a clinical or forensic toxicology laboratory (1-3). Particularly in cases where the results are relevant to legal proceedings, confirmatory tests are essential to ensure the accuracy of results. The main requirements of the confirmatory methods are specificity and the capability to separate and identify the drugs of interest and related substances. To ensure reliability and accuracy, the confirmatory tests must be different in chemical and physical principles from those of the screening tests. Immunochemical methods, such as enzyme immunoassay, fluorescence immunoassay, and radioimmunoassay, are generally preferred for screening. Unfortunately, the antibodies used in many immunoassays may cross react with chemically related drugs and metabolites. This is the main reason for the extensive application of chromatographic confirmatory tests. The current tendency is to advocate gas chromatography/mass spectrometry (GC/MS) as the method of choice for confirmation. GC/MS is highly sensitive and specific, and its ability to couple spectral data (using the "full scan" or the selected ion monitoring modes) with retention time data allows a definitive identification of drugs (4,5). Nevertheless, many laboratories are not 9 Authorto whom correspondence shouldbe addressed.
Experimental Materials All solvents used in sample extraction and in mobile phase preparation were analytical and HPLC grade, respectively (E. Merck, Darmstadt, Germany). All reagents used in sample extraction and analysis were analytical grade and were obtained from Merck, with the exception of the enzyme ~-Glucuronidase (No. G-0876), which was purchased from Sigma Chemical Company. The following pure drugs were used as standards: morphine 9 HCI, codeine 9H3PO4, nalorphine ~ HBr, benzoylecgonine ~ 4H20, 11-nor-A9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), amphetamine, methamphetamine, barbital, allobarbital, phenobarbital, butabarbital, pentobarbital, secobarbital (Division of Narcotic Drugs, United Nations, Vienna, Austria), 7-acetamidonitrazepam, 7-aminonitrazepam, 7-aminoflunitrazepam, demoxepam, desmethylchlordiazepoxide, oxazepam, n-hydroxyethylflurazepam, and desmethylflunitrazepam, desmethyldiazepam (Hoffmann-LaRoche, Basel, Switzerland). Solid-phase extraction columns, Adsorbex-Diol 100 mg, and Adsorbex-RP8 100 mg, as well as Extrelut 3 and Extrelut 20 columns, were obtained from Merck.
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Buffer solutions
Benzoylecgonine. Phosphate buffer (pH 2.1; 0.01 M) for mobile phase was prepared as follows: 0.68 g of sodium dihydrogen phosphate was dissolved in 450 mL of water; the pH was adjusted to 2.1 by addition of conc. phosphoric acid; 0.432 g of dodecyl hydrogen sulfate sodium salt was added and the resulting mixture was diluted with water to obtain a final volume of 500 mL. THC-COOH. Phosphoric acid (0.05M) for mobile phase: 3.4 mL of conc. phosphoric acid was diluted with water to obtain a final volume of 1000 mL. Amphetamines. Phosphate buffer (pH 3; 0.01M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 950 mL of water; 1.3 mL of methanesulfonic acid was added and the pH was adjusted to 3 with 5N potassium hydroxide. The resulting mixture was diluted with water to obtain a final volume of 1000 mL. Barbiturates. Phosphate buffer (pH 5.5; 0.5M): 6.8 g of potassium dihydrogen phosphate was dissolved in 95 mL of water. The resulting solution, after adjusting the pH to 5.5 with dil. phosphoric acid, was diluted with water to obtain a final volume of 100 mL. Phosphate buffer (pH 4.4; 0.01M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 980 mL of water; the pH was adjusted to 4.4 with dil. phosphoric acid and water was added to obtain a final volume of 1000 mL. Benzodiazepines. Acetate buffer (pH 5; 0.2M): The pH of 50 mL of 1M sodium acetate was adjusted to 5 by adding about 14 mL of IM hydrochloric acid and the resulting solution was diluted with water to obtain a final volume of 250 mL. Borate buffer (pH 9.2; 0.1M): 0.618 g of boric acid and 0.746 g of sodium tetraborate were dissolved in 100 mL of water. The pH of 50 mL of this solution was adjusted to 9.2 by addition of 0. IN sodium hydroxide. Phosphate buffer (pH 3.5; 0.01 M) for mobile phase: 1.361 g of potassium dihydrogen phosphate was dissolved in 950 mL of water; 1.3 mL of methanesulfonic acid was added and the pH was adjusted to 3.5 with 5N potassium hydroxide. The resuhing mixture was diluted with water to obtain a final volume of 1000 mL. Preparation of standards Concentrations of the drugs in standard solutions, used for testing the extraction procedures, were prepared by adding appropriate amounts of the stock standards to urine samples from healthy volunteers. Such stock solutions were prepared at concentrations of 10 lag/mL (except nalorphine 100 lag/mL) in methanol. These solutions were stored at 4~ in the dark and were found to be stable for at least six months. Appropriate dilutions of standard solutions, with methanol or with the mobile phases, provided working standards. Urine samples from drug users were obtained from the local hospital or from drug rehabilitation centers.
Extraction procedures Morphine. 0.5 mL of a 100-gg/mL nalorphine 9 HBr methanolic solution (internal standard) and 1 mL of conc. hydrochloric acid were added to 10 mL of urine. The mixture was hydrolyzed for 1 h at 100~ and, after cooling, 0.5 mL of a sat. ammonium sulfate solution was added. The pH was then adjusted to 9 with 25% sodium hydroxide. The solution was diluted with water to obtain a final volume of 20 mL and poured into an Extrelut 20 column. After 10 rain, elution was carried out with 40 mL of a 15% solution of isopropyl alcohol in methylene chloride. The eluate obtained was extracted twice with 3 mL of
218
0.2N hydrochloric acid. The organic phase was discarded and 0.5 mL of the sat. ammonium sulfate solution was added to the aqueous phase and the pH was adjusted to 9.2 with 25% sodium hydroxide. The mixture was again diluted with water to obtain a final volume of 20 mL and treated on a second Extrelut 20 column in the same way as on the first one. The final eluate was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 gL of HPLC mobile phase and a 20-gL aliquot was injected into the chromatograph. Benzoylecgonine. A 3.5-mL aliquot of urine was adjusted to pH 9 with 60 mg of sodium bicarbonate-potassium carbonate (2:1, w/w) and, if necessary, with small amounts of acid or base. The urine specimen was poured into an Extrelut 3 column and, after 10 min, elution was carried out with 15 mL of a 10% solution of isopropyl alcohol in chloroform. The eluate volume was reduced to approximately 100 gI_, with a stream of nitrogen at 40~ and a 5-mL aliquot of methylene chloride was added to the tube. The organic solution obtained was passed through an Adsorbex-Diol 100-mg column previously conditioned with 2 mL of methylene chloride. The column was then washed with 1 mL of methylene chloride and 4 mL of diethyl ether. Benzoylecgonine was eluted with two 500-gL aliquots of methanol. This final organic solution was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 IlL of HPLC mobile phase and a 20-1aL aliquot was injected into the chromatograph. THC-COOH. A 5-mL aliquot of urine was heated for 20 min at 60~ after adding 0.5 mL of 10N potassium hydroxide. The hydrolyzed sample was cooled to room temperature and then extracted with 10 mL of a 10% solution of ethyl acetate in nhexane. The organic phase was discarded and the remaining aqueous phase was acidified (pH 3.5) with 6N hydrochloric acid and subsequently passed through an Adsorbex RP8 100-rag column previously conditioned with 2 mL of methanol and 4 mL of water. The column was then washed with 5 mL of water and two l-mL aliquots of a 20% solution of acetone in water. THCCOOH was eluted with two 1-mL aliquots of a 10c/~' solution of ethyl acetate m n-hexane. The obtained organic solution was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 bt.L of HPLC mobile phase and a 20-btL aliquot was injected into the chromatograph. Amphetamines. The pH of a 3-mL aliquot of urine was adjusted to 11 with 10N potassium hydroxide. The sample was poured into an Extrelut 3 column, one drop 3N hydrochloric acid was poured into the collection tube and, after 10 min, elution was carried out with 15 mL of n-hexane. The eluate was evaporated to dryness under a stream of nitrogen at 35~ 1.5 mL of an 8% aqueous solution of sodium bicarbonate and 1 mL of a 0.5% aqueous solution of sodium naphthoquinone-4-sulfonate (NQS) were added to the residue. The resulting mixture was heated at 70~ for 20 min. After cooling, it was extracted with 5 mL of carbon tetrachloride and the resulting organic phase was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 uL of HPLC mobile phase and a 20-uL aliquot was injected into the chromatograph. Barbiturates. 1 mL of phosphate buffer (pH 5.5; 0.5M) was added to 2 mL of urine. The resulting mixture was poured into an Extrelut 3 column and, after 10 rain, elution was carried out with 15 mL of a 5% solution of isopropyl alcohol in methylene chloride. The eluate was evaporated to dryness under a stream of nitrogen at 40~ and the residue was reconstituted with 100 of HPLC mobile phase. 20 ,u.Lof this final solution was injected into the chromatograph.
Journal of Analytical Toxicology, Vol. 16, July/August 1992
Benzodiazepines. A 2.5-mL aliquot of urine was adjusted to pH 5 by adding dropwise dil. hydrochloric acid. 0.4 mL of acetate buffer (pH 5; 0.2M) and 0.05 mL of 13-glucuronidase were then added. The mixture was incubated for 5 h at 45~ After cooling, the hydrolyzed sample was adjusted to pH 8-9 with 25% sodium hydroxide and subsequently poured into an Extrelut 3 column. After 10 min, elution was carried out with 15 mL of a 10% solution of isopropyl alcohol in chloroform. The eluate was evaporated to dryness under a stream of nitrogen at 40~ and the residue was reconstituted with 2 mL of borate buffer (pH 9.2; 0.1M). The resulting solution was extracted twice with 3 mL of diethyl ether and the organic phase was evaporated to dryness under a stream of nitrogen at 40~ The residue was reconstituted with 100 laL of HPLC mobile phase and a 20-laL aliquot was injected into the chromatograph.
Enzyme Immunoassay Urine specimens were screened for the presence of opiates, benzoylecgonine, THC-COOH, amphetamines, barbiturates, and benzodiazepines by EMIT d.a.u, according to the manufacturer's procedures. Samples were processed using a Syva Model 1500 automatic pipetter--diluter connected to a Syva Autocarousel, a Gilford Stasar S-Ill spectrophotometer and a Syva CP-5000 Plus clinical processor. The cutoff levels set for the above assays were 300 ng/mL (except cannabinoids, 100 ng/mL) and corresponded to the concentration of the drugs in Syva's "low calibrator" solutions.
Results a n d D i s c u s s i o n Apparatus and chromatographic conditions The HPLC apparatus consisted of a Merck Hitachi L 6200 intelligent pump, L 4200 UV-VIS detector, D 2000 Chromatointegrator, and a Rheodyne 71-25 injection valve with a 20-~aL sample loop. A Hibar analytical column (Merck), 250 x 4 mm, packed with Lichrospher Si I00 (5 wn) and a Lichrocart 4-4 pre-column (Merck) packed with Lichrosorb (5 ~n) were used for morphine detection. A Hibar analytical column (Merck), 250 x 4 mm, packed with Lichrospher 100 RP8 (5 ~trn) and a Lichrocart 4-4 pre-column (Merck) packed with Lichrospher 100 RP8 (5 larn) were used for benzoylecgonine, THC-COOH, amphetamine, barbiturate, and benzodiazepine detection. The eluents were monitored at 225,233, 211,460, 212, and 234 nm, respectively, and for morphine, benzoylecgonine, THCCOOH, amphetamines, barbiturates, and benzodiazepines. All chromatographic separations were accomplished at room temperature using the following mobile phases. Morphine. n-Hexane-methylene chloride-methanol containing diethylamine (0.75%, v/v) (72.5:20:7.5, v/v/v). Flow rate: 1.35 mL/min. Benzoylecgonine. Phosphate buffer (pH 2.1; 0.01M) containing 0.003M dodecyl hydrogen sulfate sodium salt-acetonitrile (60:40, v/v). Flow rate: 1 mL/min. THC-COOH. 0.05M phosphoric acid-acetonitrile (35:65, v/v). Flow rate: 0.8 mL/min. Amphetamines. Phosphate buffer (pH 3; 0.01M) containing 0.02M methanesulfonic acid-acetoni~le (45:55, v/v). Flow rate: 1 mL/min. Barbiturates. Phosphate buffer (pH 4.4; 0.01M)-acetonitrile with the following gradient composition: t = 0 min, 70:30 v/v; t = 8 min, 60:40 v/v; t = 14 min, 60:40 v/v; t = 15 min, 70:30 v/v. Flow rate: 1 mL/min. Benzodiazepines. Phosphate buffer (pH 3.5; 0.01M) containing 0.02M methanesulfonic acid-acetonitrile, with compositions suitable for two alternative analyses. Benzodiazepine metabolites (Mobile phase 1). t = 0 min, 70:30 v/v; t = 1 min, 70:30 v/v; t = 7 min, 60:40 v/v; t = 16 min, 60:40 v/v; t = 18 min, 70:30 v/v. Flow rate: 1 mL/min. Oxazepam and desmethyldiazepam (Mobile phase 2). 55:45 v/v; Flow rate: 1 mL/min. All liquid chromatographic solvents and buffer solutions were filtered through ANODISC 47 (0.2 ~un) filters (Merck) and degassed under vacuum with stirring before use. Glassware for the THC-COOH detection was silanized before use to prevent adsorption of the drug metabolite on borosilicate glass.
Typical chromatograms obtained by HPLC analysis of the above mentioned individual drugs or classes of drugs are reported in Figures 1-6. Every figure shows the analysis of (a) a standard solution containing a single drug or a mixture of drugs, (b) an extract of a urine sample spiked with the substance(s) under analysis, and (c) an extract of a corresponding EMIT positive urine sample.
a
b
c
LL Figure 1. Liquid chromatograms of (a) a standard mixture of opiates (1-3), (b) an extract of a urine sample spiked with 1-3 (5 lag/mL each), and (c) an extract of an EMIT positive urine sample for opiates (containing 1:5 ~/mL). Peak assignments: 1 = nalorphine, 2 = codeine, and 3 = morphine.
a
b
c
Figure 2. Liquid chromatograms of (a) a standard solution of benzoylecgonine (1), (b) an extract of a urine sample spiked with 1 (1 ~/mL), and (c) an extract of an EMIT positive urine sample for cocaine metabolite.
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a
b
a ".
c
b I
r'
Figure 3. Liquid chromatograms of (a) a standard solution of 11-nor-tPtetrahydrocannabinol-9-carboxylic acid (1), (b) an extract of a urine sample spiked with 1 (0.4 lag/mL), and (c) an extract of an EMIT positive urine sample for cannabinoids.
;2~3
2
"3
c
!2 3
Figure 5. Liquid chromatograms of (a) a standard mixture of six barbiturates (1-6), (b) an extract of a urine sample spiked with 1-6 (10 lag/mL each), and (c) an extract of an EMIT positive urine sample for barbiturates. Peak assignments: 1 = barbital, 2 = allobarbital, 3 = phenobarbital, 4 = butabarbital, 5 = pentobarbital, and 6 = secobarbital.
a 1
~
"
b
r
d
b
,
e
e 9 I
1112
2
8 9 2 3
__t_T_ Figure 4. Liquid chromatograms of (a) a standard mixture of amphetamines derivatized with NQS, (b) an extract of a urine sample spiked with amphetamine and methamphetamine (1 ~g/mL each) after derivatization with NQS, and (c) an extract of an EMIT positive urine sample for amphetamines (after derivatization with NQS). Peak assignments: 1 = NQS-derivatized amphetamine and 2 = NQS-derivatized methamphetamine.
Extractions
An initial step common to all the proposed procedures consisted of the adjustment of the extraction pH. The best recoveries were obtained at pH 9 for the amphoteric compounds, morphine and benzoylecgonine, and at strong and mildly acidic pH, respectively, for THC-COOH and barbiturates. The chromatographic analyses of morphine, THC-COOH, and benzodiazepines could be efficiently performed only after a prior hydrolytic treatment of the corresponding urine specimens in order to obtain the cleavage of the relative glucuronic conjugates. In this context acidic hydrolysis was used for morphine sample treatment, although enzymatic hydrolysis can be similarly applied. Alkaline hydrolysis proved to be more efficient for THCCOOH assay with respect to cleanliness of the extracts and reaction times, according to previous papers (20-21). The application of enzymatic hydrolysis was essential for benzodiazepinic compounds because of their widely known relative instability in acidic conditions. As previously reported (18), the extensive use of the Extrelut extraction columns as an alternative to the conventional liquid-liquid extractions speeded up sample preparation (enabling more samples to be processed per day),
220
Figure 6. Liquid chromatograms of (a) a standard mixture of nine benzodiazepine metabolites (1-9) (Mobile phase 1), (b) a standard mixture of 6 and 9 (Mobile phase 2), (c) an extract of a urine sample spiked with 1-9 (1 i.tg/mL each) (Mobile phase 1 ), and (d) an extract of an EMIT positive urine sample for benzodiazepines (Mobile phase 2) Peak assignments: 1 = 7-acetamidonitrazepam, 2 = 7-aminonitrazepam, 3 = 7-aminoflunitrazepam, 4 = demoxepam, 5 = desmethylchlordiazepoxide, 6 = oxazepam, 7 = N-hydroxyethylflurazepam, 8 = desmethylflunitrazepam, and 9 = desmethyldiazepam.
eliminated incidental emulsions, and allowed high extraction recoveries to be obtained. In particular, the use of n-hexane as the liquid extraction phase resulted in the efficient extraction of amphetamines and reduced the co-extraction of polar urinary components. Similarly, for the barbiturates, the pH adjustment to 5.5 of the urine specimens avoided the extraction of interfering basic compounds and did not require additional purification steps. Solid-phase extractions were efficiently employed for the isolation of benzoylecgonine and THC-COOH. In the former case, the hydrogen bondings and dipole-dipole interactions between the analyte molecules and the diol groups on the sorbent (Adsorbex-Diol column) allowed the polar cocaine metabolite present in a nonpolar solvent of dissolution (CH2C12) to be selectively retained. In the latter case, the THC-COOH was selectively retained by the solid nonpolar sorbent (Adsorbex RP8 column) by means of hydrophobic interactions between the octyl chains of the stationary phase and the alkyl and aromatic moieties of the analyte molecules. The selectivity and specificity, important pe-
Journal of AnalyticalToxicology,Vol. 16, July/August1992
culiarities of the solid-phase extraction, were exploited with the optimization of the column washing steps: diethyl ether, on the one hand, as well as water and a 20% solution of acetone in water, on the other, removed some sample matrix interferences (salts, ionic compounds, UV-absorbing urine components, etc.), enabling relatively clean final extracts to be obtained containing the substance(s) under analysis. With respect to the proposed HPLC analyses, ultraviolet detection proved to be effective. However, a derivatization reaction was necessary for the analysis of urine samples containing amphetamines because these amines show only relatively weak absorption bands in UV range. The reaction with NQS formed highly colored derivatives of amphetamine and methamphetamine that showed strong absorption bands in UV (248-280 nm) and visible ranges (450-460 nm). HPLC analysis Reversed-phase liquid chromatography with a C 8 column was the preferred technique for the analysis of the drugs under consideration. A normal phase column was used only for morphine. In this context the capability of detecting the codeine proved to be particularly useful because of the high cross-reactivity between morphine, codeine, and their glucuronides in immunoassays. The ion-pair HPLC technique obtained the best chromatographic performances: the benzoylecgonine, the amphetamine derivatives, and the benzodiazepinic compounds, all positively charged at the acidic pH of the relative mobile phases, form neutral ion pairs with dodecyl hydrogen sulfate sodium salt or methanesulfonic acid, permitting efficient hydrophobic interactions between the analytes and the stationary phase. Mobile phases with solvent composition gradients generated fairly good chromatographic separations in barbiturate and benzodiazepine analyses. With regard to the benzodiazepine analysis, two mobile phases with different compositions were found to be useful and were Table I. Comparison between GC/MS and H P L C - U V as Methods of Confirmation for EMIT Positive Results EMIT d.a.u. assay Opiates
Positive Correlation(%) samples GC/MS HPLC-UV
Notes
125
96
92
70
98.5
97.1
102 54
98 98.2
89.2 98.1
Barbiturates
32
100
100
All samplespositive for phenobarbital
Benzodiazepines
80
92.5
87.5
2 samplescontaining benzodiazepinesnot included in the HPLC assay
Cocaine metabolite Cannabinoids Amphetamines
3 samplespositive for codeine by both confirmation techniques
4 samplespositive for methamphetamine by both confirmation techniques
consequently adopted for a general screening of various benzodiazepine metabolites and for oxazepam and desmethyldiazepam detection. The HPLC methods were developed merely to confirm the presence of drugs of abuse in urine and not to quantify them. Consequently, the use of internal standards was not considered strictly necessary. However, because the positive identification of the substances under analysis is based on retention times, a routine check of their relative daily variability is suggested. This can be accomplished by simply injecting the pure standards at regular intervals. Validation of detections The liquid chromatographic procedures showed recoveries of the substances in the range 75-85% (n -- 5, CVs < 12%). Specificity and sensitivity were optimized to allow the detection of urinary levels of the substances under analysis near the corresponding cutoff levels of the EMIT d.a.u, assays used as screening tests. The limits of detection (K -- 3) of the above mentioned HPLC procedures were as follows: 100 ng/mL morphine; 75 ng/mL benzoylecgonine; 50 ng/mL THC-COOH; 60 ng/mL amphetamine and methamphetamine, 100 ng/mL phenobarbital and 150 ng/mL secobarbital; and 100 and 200 ng/mL of oxazepam, using mobile phases 2 and 1 respectively. A consistent number of EMIT positive urine samples for the six classes of drugs of abuse were analyzed for confirmation purposes by HPLC and GC/MS procedures routinely employed in our laboratory (5). These duplicate analyses were made to evaluate the applicability of the liquid chromatographic procedures as effective analytical tools in confirmatory tests. The results (see Table I) demonstrate that the proposed HPLC procedures can offer performances comparable to those typically obtainable by the majority of GC/MS procedures. The following is a list of drugs found to be present by GC/MS in association with the reported drugs of abuse in more than 300 samples analyzed for the correlation studies. Under the chromatographic conditions applied, no interferring peaks were observed.
Drugs Found Not to Interfere with the HPLC Procedures Acetaminophen Acetylsalicylicacid Amitriptyline Buprenorphine Caffeine Carbamazepine Chlorpromazine Desipramine Dextromethorphan Doxepin Ephedrine Fenfluramine Imipramine Lidocaine Loxapine Meperidine Methadone Methaqualone Naloxone Naltrexone Nicotine
Orphenadrine Oxicodone Papaverine Pentazocine Phendimetrazine Phenmetrazine Phentermine Phenylpropanolamine Phenytoin Primidone Procaine Prometazine Propoxyphene Propyphenazone Theobromine Theophylline Trazodone Triflupromazine Trimethoprim Trimipramine
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Conclusions
The reported procedures prove the general applicability of liquid chromatography with UV detection for confirmation of drugs of abuse in urine. Especially when the sample treatment is developed to reduce the presence of interfering UV-absorbing compounds and adequate chromatographic conditions are chosen, liquid chromatography with UV detection is a valid analytical tool which merits serious consideration for specific and sensitive identifications in routine cases. Furthermore, the analytical system consisting of immunoassay screening and HPLC confirmation tests can be considered sufficiently reliable and specific to be applied in clinical and forensic toxicology laboratories for detection of drugs of abuse in urine. Nonetheless, GC/MS actually remains the ideal analytical means of confirmation of the presence of drugs in biological fluids, exhibiting generally better confirmatory power than that of other techniques, such as HPLC.
9.
10.
11.
12.
13.
14.
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