One-Step Immunoassay for Acetaminophen and Salicylate in Serum, Plasma, and Whole Blood

Transcription

1 Journal of Analytical Toxicology, Vol. 27, Seplember 2003 [ TechnicalNote One-Step Immunoassay for Acetaminophen and Salicylate in Serum, Plasma, and Whole Blood Wu Song* and Chao Dou Applied Biotech, Inc., San Diego, California Abstract Acetaminophen (APAP, n-acetyl-p-aminophenol) and salicylate (ASA, acetylsalicylic acid) are two of the most frequently overdosed drugs reported in the emergency room. Most APAP and ASA tests are performed with analytical instruments in laboratories, which are not always ideal for emergency situations. A rapid membrane and gold particle-based immunoassay for the simultaneous detection of APAP and ASA has been developed. Validation studies verified the cutoffs for the assay as 25 pg/ml for APAP and 100 pg/mt for ASA, respectively, and the accuracy study indicated that the agreement between the APAP and the ASA test and reference methods is 99%. One hundred thirly-three structurally unrelated chemicals and metabolites that may be present in human blood specimens were tested and found not to cross-react with the test. Introduction Acetaminophen (APAP, n-acetyl-p-aminophenol) and salicylate (ASA, acetylsalicylic acid) are two of the most frequently used analgesic/antipyretic drugs. Each year in the United States over 111,000 APAP overdose cases are reported to poison control centers, with 40,000 associated emergency department cases (1). The therapeutic concentration of APAP is ljg/ml (2). After ingestion, more than 90% of APAP is metabolized through conjugation to glucuronate or sulfate in the liver, a nontoxic route. The remaining 10% is oxidized within the liver to a highly toxic free-radical form, N-acetyl-benzoquinone imine (3). Serum APAP levels greater than 200 IJg/mL at 4 h postdosage or 50 IJg/mL at 12 h post-dosage are associated with serious liver damage (4,5). In the case of an overdose, the toxic metabolite accumulates in liver cells and eventually causes hepatic cell death. Acute APAP toxicity shows few early signs, and liver damage is not clinically evident until 12 h or more 9 Author ~o whom correspondence should be addressed: Wu Song, M.D., Ph.D., Research Scientist, R&D Department, Applied Biotech, Inc., t 0237 Flanders Court, San Diego, CA swu@abiapogen[.corn. after ingestion (6,7). ASA is a common drug used in many formulations because of its analgesic and anti-inflammatory properties. ASA is quickly metabolized into salicylate after ingestion and is eliminated by conjugation with glycine and glucuronic acid (7). The therapeutic concentration of ASA is pg/ml (2). In severe poisoning, the blood ASA concentration may exceed 7001Jg/mL (8). Some mild toxicity can occur with patients on long-term, high-dose aspirin therapies. Determination of APAP and ASA overdose is very important in order for doctors to properly treat patients in an emergency situation. Currently, quantitative methods are widely used in the hospitals. Although these tests are accurate, they require significant instrumentation to perform the tests. To reduce assay turnaround time, we have developed a rapid lateral flow immunoassay for simultaneous detection of APAP and ASA in human serum, plasma, or whole blood. Our results present the performance characteristics of the test including its analytical sensitivity, accuracy, specificity, and potential interference from chemicals that can be found in human blood. Materials and Methods Acetaminophen/salicylate test device The acetaminophen/salicylate test device was manufactured by Applied Biotech, Inc. (San Diego, CA). Chemicals All reagents, chemicals, APAP, and ASA diagnostic kits were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Specimen collection and storage The venipunctured whole blood (EDTA, citrate, or heparin anticoagulated), plasma, and serum were used for testing immediately or stored at 2-8~ for up to three days before use. Principles and procedure of lateral-flow immunoassay The APAP/ASA test is a lateral-flow immunoassay. The test 366 Reproduction (pholocopyin 8) of editorial content of this journal is prohibited without publisher's permission.

2 device contains a membrane strip that has been precoated with APAP- and ASA-protein conjugates on the test line regions. Colored anti-apap/asa antibody-colloidal gold particles were placed on a pad at the end of the membrane. In the absence of the drugs in the test specimen, the colored antibody gold particles migrate with the sample solution by capillary action across the membrane to the test regions, forming visible lines as the antibody and drug conjugate complex. Therefore, formation of visible lines in the specific test areas occurs when the test specimen is free of drugs. When either drug is present in the test specimen, the drug in the specimen competes with the drug conjugate on the test band region for the limited antibody binding sites, preventing the formation of the test line. Therefore, absence of a colored line on the test region indicates a positive result for that particular drug. A control line with a different antigen/antibody reaction is also added to the membrane strip to indicate that a sufficient volume of specimen has been added and to ensure proper flow was obtained. This control line should always appear regardless of the presence of APAP/ASA. The test was performed according to the following procedure: a sample of 40 pl of serum or plasma or 80 pl of whole blood was added into the well. Once the sample was absorbed, 160 pl of dilution buffer was added. The test result was recorded between 5 and 8 min after the addition of dilution buffer. Sigma APAP kit procedure After removal of interfering proteins from serum or plasma with trichloroacetic acid, APAP was reacted with nitrous acid to form a nitro-derivative, which assumed a deep yellow color in alkaline medium. The intensity of the yellow color measured at 430 nm was proportional to the APAP concentration in the test sample. The test was performed according to the manufacture's package insert (9). Sigma ASA kit procedure The test is based on the formation of a color compound between ASA and Fe The difference in absorbance reading at 540 nm, before and after addition of ferric ions, was proportional to ASA concentration in the test specimen. The test was performed according to the manufacturer's package insert (10). Cutoff verification The cutoff values of the APAP/ASA test were evaluated by testing spiked serum samples. APAP and ASA were separately spiked into drug-free serum at target concentrations equal to 200%, 150%, 125%, 75%, 50%, 25%, and 0% of the cutoff values. The concentrations of APAP and ASA in the spiked samples were measured by Sigma APAP/ASA kits. Forty spiked serum samples at each concentration level were tested with APAP/ASA devices. Accuracy study APAP and ASA were separately spiked into serum samples (V.A. Hospital San Diego, CA) at various concentrations. The concentrations of APAP or ASA were measured by the Sigma kits. Blind-labeled spiked-serum samples were tested at three point-of-care sites and at Applied Biotech, Inc. Reproducibility study APAP and ASA were spiked into serum at six different concentrations [APAPIASA: 010 pg/ml (0% of the assay cutoff), pg ImL (50% of the assay cutoff), pglml (75% of the assay cutoff), pglml (125% of the assay cutoff), Table I. Cutoff Value Determination of the APAP and ASA Tests Number Number Percentage Number of positive of negative of correct Concentration of tests results results results APAP 0 pg/ml % 6.25 IJg/mL % 12.5 pglml % 18.8 pg/ml % 25 pglml % ijg/ml % 37.5 pg/ml % 43.8 pg/ml % 50 pglml % ASA 0 pglml % 25 IJg/mL % 50 IJg/mL % 75 pglml % I00 IJglml % 125 pglml I 97.5% 150 pglml % 175 pg/ml % 200 IJg/mL % Table II. Accuracy Study of the APAP/ASA Test Concentration range (pg/ml) determined by Sigma APAP KIT APAP Test Positive Concentration range (pg/ml) determined by Sigma ASA KIT ASA Test Positive (0-75% cutoff) (76-100% of cutoff) ( % of cutoff) ( % of cutoff) (0-75% cutoff) I 00 (76-100% of cutoff) I 25 (I 01-I 25% of cutoff) (I % of cutoff)

3 pg/ml (150% of the assay cutoff), and 50/200 pg/ml (200% of the assay cutoff)]. At each site, 18 tests were run each day for 3 consecutive days. In all, 216 tests were performed at 4 sites. The reproducibility of the APAP/ASA test was evaluated at four sites, including three point-of-care sites. Specificity The specificity of APAP/ASA test was determined by evaluating the cross-reaction of the test with various chemicals. Each compound tested was spiked into methanol or phosphatebuffered saline buffer at a concentration of 2 mg/ml and Table III. Cross-Reaction of Structurally Related Compounds on the APAP and ASA Tests* NHCOCH 3 I Journal of Analytical Toxicology, Vol. 27, September 2003 diluted with drug-free serum to a concentration of 100 and 10 pg/ml, respectively. The prepared solutions were then used to evaluate the cross-reaction of the APAP/ASA test device. Any sample yielding a positive result was serially diluted and retested to determine the threshold concentration at which the compound cross-reacted. Anticoagulant interference study Individual blood samples, collected in EDTA, citrate, or heparin anticoagulant tubes, were purchased from the Red Cross (San Diego, CA). APAP and ASA were separately spiked into the blood samples at various concentrations. After the determination of APAP or ASA concentrations by the Sigma APAP/ASA kits, the spiked whole blood samples were tested by the one-step APAP/ASA test. A. Test compounds [O2 Acetaminophen Substitute group on benzene ring Acetaminophen NHCOCH 3 Acetophenetidin NHCOCH 3 OC2Hs 4-Aminophenol NH 2 Aniline NH2 Hippuric acid CONC o-hydroxyhippuric acid CONC p-benzoquinone O O B. Test compounds Salicylic acid Aspirin p-aminosalicylic acid 3-Aminosalicylic acid 5-Aminosalicylic acid 3-Methylsalicylic acid Gentistic acid Diflunisal Salsalate I I 6 s Salicylic Acid * (A) APAP test and (B) ASA Test. Substitute group on benzene ring OOCCH 3 OOC-C6H6- NH2 CH3 NH 2 Detection level (pg/ml) 25 Detection level 5 (pg/mt) NH2 F-C6H6-F Results Cutoff value The test was designed to have cutoff values for APAP and ASA at 25 and 100 pg/ml, respectively. At one level below the cutoff concentration (50% of the cutoff), the majority of the samples tested should give true-negative results; at one level above the cutoff concentration (200% of the cutoff), the majority of the samples tested should give true-positive results. The results of the cutoff verification for APAP/ASA test are summarized in the Table I. All samples with APAP concentrations equal to or below 12.5 IJg/mL and all samples with ASA concentrations equal to or below 50 pg/ml (50% of the cutoff) were negative. All the samples with APAP concentrations equal to or above 37.5 pg/ml and all samples with ASA concentrations equal to or above 150 pg/ml (150% of the cutoff) were positive. Based on these results, the cutoff values of APAP and ASA test were determined to be 25 and 100 pg/ml, respectively. Accuracy The results of the accuracy study are summarized in Table II. Twelve of 12 samples with APAP concentrations ranging from 0% to 75% of the cutoff value and 12 of 12 samples with APAP concentrations ranging from 76% to 100% of the cutoff value were true-negative. Among 15 samples with APAP concentrations from 104% to 124% of the cutoff value, 1 was false-negative and 14 were true-positive. All 99 samples with APAP concentrations higher than 244% of the cutoff value were true-positive. Thirty-nine of 39 samples with ASA con- 368

4 centrations ranging from 0% to 75% of the cutoff value were true-negative. Among 11 samples with ASA concentrations from 76% to 100% of the cutoff value, 9 samples were true-negative, and 2 samples were false-positive. Sixteen of 16 samples with ASA concentrations from 101% to 125% of the cutoff value were true-positive. Ninety-nine of 99 samples with ASA concentrations higher than 126% of the cutoff value were true-positive. Based on these results, it is conclude, that the APAP/ASA test correctly detects APAP and ASA in Table IV. Cross-Reaction of Structurally Unrelated Compounds on the APAP/ASA Test Albumin Dopamine Naloxone Amitriptyline Doxepin Naltrexone Amobarbital Doxylamine Naphthaleneacetic-~ acid Amoxapine Ecgonine Naproxen-[+] Amphetamine-o Ecgonine methyl ester Nicotine-[-] Amphetamine-t. EDDP Nicotinic acid Ampicillin Ephedrine-[ Norephedrine-[ Aspartame Ephedrine-N-methyl-(1 R,2S)- Nortriptyline Atropine [-] Noscapine hydrochloride Baclofen Epinehrine-[ Ofloxacin Benzocaine Erythromycin Orphenadrine Benzoylecgonine Famprofazone Oxalic acid Bilirubin Fenofibrate Oxazepam Brompheniramine-[+] Fluoxetine Oxycodone Bupivacaine Furosemide Penicillin-G Buspirone Gemfibrozil Pentobarbital Butabarbital Glucose Perphenazine Caffeine Guaiacol glyceryl ether Phencyclidine Carbamazepine Hemoglobin Phenelzine Carisoprodol Hematropine-D,t Pheniramine Chlordiazepoxide Hydrochlorothiazide Phenobarbital Chloroquine Hydrocodone Phenolhiazine Chlorpheniramine-[ Hydromorphone Phentermine Chlorpromazine Ibuprofen Phenylelhylamine-L Chlorprothixene Imipramine Phenylethylamine-~ Chlorthalidone Isoproterenol-[ Primidone Clobazam Ketamine Procaine Clofibrate Lidocaine Promazine Cocaine MDMA-[ Promethazine Codeine Mebeverine Propoxyphene-D Creatine Mephentermine Protriptyline Creatinine Maprotiline Pseudoephedrine Cyclobenzaprine Meperidine Quinidine Cyclodextrin-'l Mes Quinine Cyproheptadine Methadol Riboflavin Dantrolene Methadone Ritodrine Delorazepam Methamphetamine Ranitidine Deoxyephedrine-[-] Methapyrilene Secobarbital Dexamethasone Methaqualone Sodium chloride Dextromethorphan Methylenedioxyamphetamine- Sulindac Diazepam [ (MDA) Thioridazine Dicyclomine Methylphenidate Trehalose Dimethylaminoantipyrine-4 Metoclopramide Trifluoperazine Diphenhydramine Morphine Tyramine Diphenylhydantoin-5,5 Morphine-3-~D-glucuronide Vitamin C human serum, plasma, and whole blood specimens at the stated cutoff levels. Specificity Cross-reactivity of the APAP/ASA test with chemicals structurally related to acetaminophen is summarized in Table III. Acetaminophen, which has an NHCOCH3 group at position I and an group at position 4, reacted at a level of 25 pg/ml. Substitution of the two groups with other functional groups alters the cross-reactivity of the test significantly. For example, acetophenetidin with OC2Hs group at position 4 and 4-aminophenol with NH2 group at position 1, were nonreactive with the assay when tested at a level of 100 lag/me Compounds such as aniline, hippuric acid, o- hydroxyhippuric acid, and p-benzoquinone, which have groups other than NHCOCH3 at position 1, were also negative when tested at a level of 100 pg/ml. Cross-reactivity of the APAP/ASA test with chemicals structurally related to acetylsalicylic acid is summarized in Table IIIB. Salicylic acid, which has a group at position i and an group at position 2, was positive IJg/mL. Aspirin, which has a group at position 1 and an OOCCH 3 group at position 2, was positive at 50 1Jg/mL. p-aminosalicylic acid, which has a group at 1, an group at 2, and an NH2 group at position 4, was positive at 12.5 pg/ml. However, 3- and 5-salicylate derivatives such as 3-aminosalicylic acid, 5-aminosalicylic acid, 3-methylsalicylic acid, gentistic acid, and diflunisal were negative IJg/mL. Additionally, salsalate, with an --OOC-C6H6- group instead of an group at position 2, was negative pg/ml These results indicate that both APAP and ASA tests are specific for the respective compounds. Table IV summarizes the cross-reactivity of structurally unrelated compounds on the APAP/ASA test. All of the chemicals, most of which are drugs and drug metabolites, were found to be negative when tested at concentrations of 10 and 100 IJg/mL, respectively. Anticoagulant interference study The results of the anticoagulant interference study are presented in Table V. When APAP and ASA were spiked into EDTA anticoagulated whole blood samples, 4 of 4 samples with APAP concentrations from 76% to 80% of the cutoff value were true negative. Ten of 10 samples with APAP concentrations from 136% to 272% of the cutoff value were true positive. Four of 4 samples with ASA concentrations from 41% to 88% of the cutoff value were true negative. One of 369

5 Table V. Anticoagulant Interference Study of the APAP/ASA Test APAP test ASA test Concentration range Anticoagulant (pg/ml) Positive Positive EDTA (76--80% cutoff) ( % cutoff) (41--88%cutoff) 93 (93% cutoff) ( % cutoff) Citrate (48-64% cutoff) ( % cutoff) (42-85% cutoff) ( % cutoff) Heparin i 8-24 (72-96% cutoff) ( % cutoff) (8-84% cutoff) ( % cutoff) 1 sample with an ASA concentration at 93% of the cutoff value was false positive. Fourteen of 14 samples with ASA concentrations from 120% to 430% of the cutoffvalue were true positive. When APAP and ASA were spiked in the citrate anticoagulated whole blood samples, 4 of 4 samples with APAP concentrations from 48-64% of the cutoff value were true positive. Nine of 9 samples with APAP concentrations from 124% to 228% of the cutoff value were true positive. Twelve of 12 samples with ASA concentrations from 42% to 85% of the cutoff value were truenegative. Fourteen of 14 samples with ASA concentrations from 101% to 204% of the cutoff value were true positive. When APAP and ASA were spiked in the heparin anticoagulated whole blood samples, 4 of 4 samples with APAP concentrations from 72% to 96% of the cutoff value were true negative. Six of 6 samples with APAP concentrations from 108% to 204% of the cutoff value were true positive. Four of 4 samples with ASA concentrations from 8% to 84% of the cutoff value were true negative. Two of 2 samples with ASA concentrations from 220% to 231% of the cutoff value were true positive. From these results, it is concluded that there is no interference effect on the APAP/ASA test when using EDTA, citrate, or heparin as an anticoagulant APAP at 50 IJg/mL, the device cutoff value should be set at 25 IJg/mL. As demonstrated in the cutoff validation study, the APAP test can detect 100% of the samples with an APAP concentration at a level greater than 50 IJg/mL. For the ASA test, a low cutoff concentration at 100 ljg/ml was selected to ensure the detection of potential toxic concentrations in patients whose ingestion history might not have been immediate (11). The cutoff values of the APAP/ASA test were validated by the cutoff verification study. The accuracy study results demonstrated that the agreement between the lateral-flow 14 immunoassay and the reference methods is 99% for both APAP and ASA tests. These re- 0 sults indicate that the lateral flow ira- 2 munoassay for APAP and ASA can accurately detect these drugs in serum samples. The results of the cross-reactivity testing demonstrate that both APAP and ASA tests are specific for the respective compounds. Neither the APAP nor ASA assays were found to cross-react with 133 structurally unrelated drugs and metabolites I~g/mL. At normal dosage, the blood level of these drugs and metabolites should not exceed 100 IJg/mL. Different anticoagulants in human whole blood samples were also tested; none were found to interfere with the performance of the device. Compared with Sigma APAP and ASA tests, which are colorimetric assays, the lateral-flow immunoassay provides the test result more quickly. It should be suitable for emergency room use because the test saves time and enables physicians to make an accurate decision in diagnosing suspected overdose patients and, thus, reducing the cost for patient care. Acknowledgments The authors wish to thank their colleagues in the DOA group for their assistance and support. Sincere thanks are extended to Dr. James Tung for his great advice. Discussion Lateral-flow immunoassays have been widely used and accepted for the detection of human chorionic gonadotropin and H. pylori in human blood samples in clinical settings. These tests offer advantages of being rapid and user friendly. This is the first time that this type of lateral-flow test has been used to detect APAP or ASA drug overdose in human serum, plasma, and whole blood samples. The cutoff value determined is the threshold of detection. The concentration range between the visible and invisible line in a lateral-flow immunoassay can be + 50%. For example, to detect References 1. A. Wu, L. Broussard, R. Hoffman, T. Kwong, C. McKay, T. Moyer, E. Otten, S. Welch, and P. Wax. National Academy of Clinical Biochemistry Laboratory Medicine practice guidelines recommendations part IV. Recommendations on laboratory assays for other toxicants as causes of poisonings. Clin. Chem. 49: (2003). 2. W. Robert. Evaluating acetaminophen and salicylate poisoning in an emergency setting. Lab. Med. 29:33-37 (1998). 3. G. Bray. Liver failure induced by paracetamoi. Br. Med. J. 306: (1993). 4. B.H. Rumack and R.G. Peterson. Acetaminophen overdose: incidence, diagnosis, and management in 416 patients. Pediatrics 62: (1978). 5. H. Mathew and A.A.H. Lawson. Treatment of common acutepoi- 370

6 sonings, 4th ed. Churchill Livingston, New York, NY, 1979, p T.J. Meredith and R. Goulding. Oaracetamol. Postgrad. Med. 56" (1980). 7. S.J. Atwood. The laboratory in the diagnosis and management of acetaminophen and salicylate intoxication. Pediatr. Clin. North Am. 27: (1980). 8. C. Higgins. Measurement of aspirin and paracetamol metabolites. Nursing Ti'mes 8:40-41 (1996). 9. Sigma Diagnostics Acetaminophen (Procedure No. 430), Revised July Sigma Diagnostics, St. Louis, MO, catalog number 430-A. 10. Sigma Diagnostics Salicylate (Procedure No. 530), Revised April Sigma Diagnostics, St. Louis, MO, catalog number 530A. 11. A.K. Done. Salicylate, pharmacokinetics, and the pediatrician. Pediatrics 54: (1974) Manuscript received January I0, 2002; revision received December 2,