Roger L. Bertholf

Detection of Cocaine and Its Metabolites in Breast Milk

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

False-Positive Acetaminophen Results in a Hyperbilirubinemic Patient

BACKGROUND Acetaminophen was falsely detected in the plasma of a severely jaundiced patient, and a methodologic interference from bilirubin was suspected. METHODS Acetaminophen was measured by an enzymatic method (GDS Diagnostics). The putative bilirubin interference was investigated in 12 hyperbilirubinemic specimens and in bilirubin linearity calibrators. The analytical method was modified to correct for background absorbance at a second wavelength. Hyperbilirubinemic specimens were fortified with acetaminophen to assess the effect of the interference on acetaminophen measurements. RESULTS Acetaminophen was detected in 12 specimens from hyperbilirubinemic patients without a history of recent acetaminophen exposure. Dilution of hyperbilirubinemic specimens produced a nonproportional decrease in apparent acetaminophen concentrations, and no acetaminophen was detected when bilirubin was <50 mg/L. Background correction at a second wavelength failed to compensate for the interference. Although erroneous acetaminophen concentrations were detected in all specimens with high bilirubin, acetaminophen measurements in fortified specimens were accurate. CONCLUSION The data are consistent with bilirubin interference in the enzymatic and/or chromogenic reactions involved in the acetaminophen method.

Use of urine cotinine to validate smoking self-reports in U.S. Navy recruits

Cotinine analysis of urine has been used by many researchers to determine validity of smoking self-reports. This technique is easy and inexpensive, but has not been used previously in military smoking studies. This study incorporated a random validation of self-reported smoking by U.S. Navy recruits participating in a smoking relapse program (N = 496). Results of cotinine analysis indicate only a 1% misrepresentation of actual smoking status. These results suggest smoking self-reports from U.S. Navy recruits are very good indicators of actual smoking status.

Human chorionic gonadotropin in cervicovaginal secretion as a predictor of preterm delivery.

Background: Preterm delivery is one of the important problems in obstetrics and finding a way for its prediction and prevention has always been under investigation. Materials and Methods: This study carried out to determine whether human chorionic gonadotropin (HCG) detected in cervicovaginal secretion of patients with symptoms suggestive of preterm labor is a predictor of preterm birth, and to determine the cut-off values for HCG in prediction of delivery before 37 weeks of pregnancy. 150 pregnant women with gestational age 24-34 weeks and diagnosis of preterm labor and intact membrane were enrolled to the study. The patients were allocated in two groups and HCG in cervicovaginal secretion was measured in all of them. The patients were followed until their delivery and were divided in two groups. Seventy one cases delivered after 37 weeks of pregnancy (term) and 79 before 37 weeks (preterm). The amount of HCG in cervicovaginal secretion of the two groups was compared. Results: Mean concentration of HCG in cervicovaginal secretion in term group was 7.9 ± 34.1 miu/ml and in preterm group 61.12 ± 66.84 miu/ml which was statistically significant (p Conclusion: Quantitative HCG concentration measurement from cervicovaginal secretions may be a useful predictor of preterm birth in symptomatic patients. This test has the advantage of low cost and wide availability.

Unexpected Urine Drug Testing Results in a Hospice Patient on High-Dose Morphine Therapy

A 41-year-old African-American woman was admitted to an inpatient hospice facility with advanced, inoperable cervical cancer. The patient was experiencing severe pain secondary to extensive local tumor invasion, osseous pelvic metastases, and sacral decubitus ulcers. Her pain was treated with an escalating-dose schedule of morphine sulfate until satisfactory analgesia was achieved with stable doses of a combination of controlled-release morphine sulfate (MSContin®, Purdue Pharma LP) 400 mg orally every 8 h, and immediate-release morphine sulfate (MSIR®, Purdue Pharma LP), 180 mg orally every 4 h, as needed for breakthrough pain (average 2 to 3 doses per day). The patient experienced several episodes of life-threatening vaginal bleeding for which she was hospitalized for red blood cell transfusions and bilateral hypogastric artery embolizations. She spent the final 12 weeks of her life exclusively on the inpatient hospice unit. Approximately 3 weeks before her death, the patient underwent urine specimen collection and analysis of morphine and metabolites. GC-MS analysis revealed the presence of morphine as well as small quantities of hydromorphone. During the past 2 decades, chronic opioid analgesic therapy (COAT) for chronic nonmalignant pain has gained increasing clinical acceptance. An unintended consequence of more liberal opioid prescription practices has been a dramatic increase in the abuse and diversion of these drugs. According the most recent National Survey on Drug Abuse and Health (1), the number of new, past-year abusers of prescription opioids was 2 147 000—more than the number of new abusers of any other single class …

Validation of a pre-existing formula to calculate the contribution of ethanol to the osmolar gap.

Purpose. The aim of this study was to validate the formula derived by Purssell et al. that relates blood ethanol concentration to the osmolar gap and determine the best coefficient for use in the formula. The osmolar gap is often used to help diagnose toxic alcohol poisoning when direct measurements are not available. Methodology. Part I of the study consisted of a retrospective review of 603 emergency department patients who had a concurrent ethanol, basic metabolic panel and a serum osmolality results available. Estimated osmolarity (excluding ethanol) was calculated using a standard formula. The osmolar gap was determined by subtracting estimated osmolarity from the actual osmolality measured by freezing point depression. The relationship between the osmolar gap and the measured ethanol concentration was assessed by linear regression analysis. In Part II of this study, predetermined amounts of ethanol were added to aliquots of plasma and the estimated and calculated osmolarities were subjected to linear regression analysis. Results. In the cases of 603 patients included in Part I of the study, the median ethanol concentration in these patients was 166 mg/dL (Q1: 90, Q3: 254) and the range ethanol concentrations was 10–644 mg/dL. The mean serum osmolality was 338 mOsm/kg (SD: 30) and a range of 244–450 mOsm/kg. The mean osmolar gap was 47 (SD: 29) and a range of − 15 to 55. There was a significant proportional relationship between ethanol concentration and osmolar gap (r2 = 0.9882). The slope of the linear regression line was 0.2498 (95% CI: 0.2472–0.2524). The slope of the linear regression line derived from the data in Part II of the study was 0.2445 (95% CI: 0.2410–0.2480). Conclusions. The results of our study are in fairly close agreement with previous studies that used smaller samples and suggest that an accurate conversion factor for estimating the contribution of ethanol to the osmolar gap is [Ethanol (mg/dL)]/4.0.

Sensitivity of an Opiate Immunoassay for Detecting Hydrocodone and Hydromorphone in Urine from a Clinical Population: Analysis of Subthreshold Results

Urine drug testing (UDT) is an emerging standard of care in the evaluation and treatment of chronic non-cancer pain patients with opioid analgesics. UDT may be used both to verify adherence with the opioid analgesic regimen and to monitor abstinence from non-prescribed or illicit controlled substances. In the former scenario, it is vital to determine whether the drug is present in the urine, even at low concentrations, because failure to detect the drug may lead to accusations of opioid abuse or diversion. Opiate immunoassays typically are developed to detect morphine and are most sensitive to morphine and codeine. Although many opiate immunoassays also detect hydrocodone (HC) and/or hydromorphone (HM), sensitivities for these analytes are often much lower, increasing the possibility of negative screening results when the drug is present in the urine. We selected 112 urine specimens from patients who had been prescribed HC or hydromorphone but were presumptive negative by the Roche Online DAT Opiate II™ urine drug screening assay, which is calibrated to 300 ng/mL morphine. Using a GC/MS confirmatory method with a detection limit of 50 ng/mL both for HC and for HM, one or both of these opiates were detected in 81 (72.3%) of the urine specimens. Examination of the raw data from these presumptive negative opiate screens revealed that, in many cases, the turbidity signal was greater than the signal obtained for the negative control, but less than the signal for the 300 ng/mL (morphine) threshold calibrator. A receiver operating characteristic curve generated for the reciprocal of the ratio of turbidity measurements in the patient specimens and negative (drug-free) controls, against the presence or absence of HC and/or HM by confirmatory analyses, produced an area under the curve of 0.910. We conclude that this opiate immunoassay has sufficient sensitivity to detect HC and/or HM in some urine specimens that screen presumptive negative for these commonly prescribed opiates at the established threshold.

“Practical Guide” to Urine Drug Screening Clarified

To the Editor: Moeller et al recently provided a timely and important review of urine drug screening. Drug abuse is a serious medical and social problem in the United States. Urine drug testing (UDT) to detect abuse and diversion of prescription controlled medications, as well as abuse of illicit substances, is increasingly important in clinical medicine. Physicians’ ability to accurately interpret UDT results, however, is poor. Education is critical; equally critical is the dissemination of accurate information. We would like to address several inaccuracies in the review. Opioids. The authors correctly assert that fentanyl and oxycodone are undetectable by most urine screens for opiate drugs, but the reasons they provide are incorrect. Fentanyl is undetectable not because it has no metabolites (it does), but because the chemical structures of fentanyl and its metabolites differ radically from those of opiates (ie, morphine and codeine). Oxycodone is generally undetectable not because it is derived from thebaine—indeed, thebaine is a precursor to both codeine and morphine in the opium poppy—but chiefly because of a minor structural difference from opiates: a 14hydroxyl group that prevents it from cross-reacting with opiate antibodies in screening assays. The authors state that semisynthetic derivatives of morphine are not used therapeutically because of their abuse potential. In fact, hydrocodone, oxycodone, and hydromorphone, all of which are listed in Table 4 as semisynthetic opiate derivatives, are among the most commonly prescribed opioids. The authors state, “Positive results for heroin abuse are caused by use of prescribed opiates, such as codeine and hydrocodone....” This is a misstatement. Codeine, morphine, (usually) hydrocodone, and heroin all yield positive opiate screens, but this means only that an individual has been exposed to an opiate. Exposure to heroin can be established only by demonstration of the 6-monoacetyl morphine metabolite by a specific confirmatory assay. Cannabinoids. The authors perpetuate outdated information that nonsteroidal anti-inflammatory drugs (NSAIDs) can produce false-positive results for cannabinoids on the Syva EMIT and other immunoassay systems. While this was once true (their reference is nearly 20 years old), Syva has solved this problem by altering the formulation of EMIT. This was never a problem for other immunoassays. The authors also state that hemp-containing foodstuffs can produce positive screens for cannabinoids. Again, while this was once true, a 2003 US Drug Enforcement Agency (DEA) ruling classified food and beverages containing any amount of tetrahydrocannabinol (THC) as Schedule I controlled substances, making it unlawful to manufacture, distribute, dispense, or import any such product without registration. This had an immediate effect on domestic manufacturers, whose hemp-containing products are now virtually free of THC. Cocaine. Until several years ago, “Health Inca Tea,” a decocanized product that indeed contained detectable quantities of cocaine, was commercially available in the United States. The importation of this and similar products has since been banned by the DEA. Whereas these products are available over the Internet, they are classified as Schedule II drugs, making illegal their importation and possession. Amphetamine and Methamphetamine. Screening for amphetamine—a term that ordinarily refers to both amphetamine and methamphetamine—by immunochemical methods has always been problematic because the phenylethylamine drug class, with its various controlled and over-the-counter derivatives, provides minimal antigenic character. Table 3 lists methamphetamine among the causes of false-positive results, but in fact amphetamine immunoassays are intended to detect both amphetamine and methamphetamine. The lenantiomer of methamphetamine, desoxyephedrine, is not a controlled drug and can produce a positive screening result, although most modern immunoassays have a considerable degree of stereoselectivity and are much less reactive with the l-enantiomer. The authors correctly note that gas chromatography–mass spectrometry (GC-MS), without modification to separate chiral compounds, cannot distinguish between controlled (and frequently abused) d-methamphetamine and its less pharmacologically active enantiomer, desoxyephedrine. However, specifications for workplace drug testing from the Substance Abuse and Mental Health Services Administration have established a standard for GC-MS confirmation of dmethamphetamine that requires the presence (at >200 ng/mL) of the metabolically demethylated product, amphetamine, since the l-enantiomer undergoes only minimal demethylation. Although there is a reference to this requirement in a footnote to Table 1, it is not explained in the discussion, leaving the impression that confirmatory testing by nonchiral GC-MS does not rule out desoxyephedrine use, when in fact it does if performed and interpreted properly. Appropriate interpretation of positive and negative results from urine drug screens often requires consideration of many variables, a point that Moeller and colleagues’ review underscores. Misinformation, however, serves only to complicate the already difficult task that physicians face when using UDT as part of their clinical practice. We strongly urge clinicians to consult with a qualified professional (eg, certified Medical Review Officer or toxicologist) when questions arise about the meaning of results from UDT.

Chromatographic methods in clinical chemistry and toxicology

Preface. List of Contributors. 1. Quality Assurance, Quality Control and Method Validation in Chromatographic Applications (Michele L. Merves and Bruce A. Goldberger). 1.1 Introduction. 1.2 History. 1.3 Definition of Quality Assurance and Quality Control. 1.4 Professional Organizations. 1.5 Internal Quality Assurance and Control. 1.6 External Quality Assurance. References. 2. Liquid Chromatographic-Mass Spectrometric Measurement of Anabolic Steroids (Don H. Catlin, Yu-Chen Chang, Borislav Starcevic and Caroline K. Hatton). 2.1 Introduction. 2.2 LC-MS Analysis of Synthetic Steroids or Animal Samples. 2.3 LC-MS Analysis of Natural Androgens in Human Samples. 2.4 Conclusion. References. 3. High-performance Liquid Chromatography in the Analysis of Active Ingredients in Herbal Nutritional Supplements (Amitava Dasgupta). 3.1 Introduction. 3.2 St JohnaAAs Wort. 3.3 Herbal Supplements with Digoxin-like Immunoreactivity. 3.4 Herbal Remedies and Abnormal Liver Function Tests. 3.5 Ginkgo Biloba. 3.6 Echinacea. 3.7 Valerian. 3.8 Feverfew. 3.9 Garlic. 3.10 Ephedra (Ma Huang) and Related Drugs. 3.11 Conclusions. References. 4. Measurement of Plasma L-DOPA and L-Tyrosine by High-Performance Liquid Chromatography as a Tumor Marker in Melanoma (Thierry Le Bricon, Sabine Letellier, Konstantin Stoitchkov and Jean-Pierre Garnier). 4.1 Introduction. 4.2 Melanogenesis. 4.3 L-DOPA Alone. 4.4 L-DOPA/L-Tyrosine Ratio. 4.5 Conclusion. References. 5. Hypersensitive Measurement of Proteins by Capillary Isoelectric Focusing and Liquid Chromatography-Mass Spectrometry (Feng Zhou and Murray Johnston). 5.1 Introduction. 5.2 A Robust CIEF-RPLC Interface. 5.3 First-Generation CIEF-RPLC-MS System for Proteins. 5.4 Second-Generation CIEF-RPLC-MS System. 5.5 Future Improvements. Acknowledgment. References. 6. Chromatographic Measurement of Transferrin Glycoforms for Detecting Alcohol Abuse and Congenital Disorders of Glycosylation (Anders Helander). 6.1 Introduction. 6.2 Transferrin Microheterogeneity. 6.3 Carbohydrate-deficient Transferrin (CDT). 6.4 Congenital Disorders of Glycosylation (CDG). 6.5 Analytical Methods for Transferrin Microheterogeneity. 6.6 Chromatographic Methods for CDT. 6.7 Chromatographic Methods for CDG. 6.8 Summary and Conclusions. References. 7. Chromatographic Measurements of Catecholamines and Metanephrines (Eric C. Y. Chan and Paul C. L. Ho). 7.1 Background. 7.2 Analytical Measurements of Catecholamines and Metanephrines. 7.3 Early Methods. 7.4 Current Chromatographic Methods. 7.5 Practical Considerations for the Stability of Urinary Catecholamines and Metanephrines During Storage. 7.6 Future Developments. Dedication. References. 8. Chromatographic Measurement of Volatile Organic Compounds (VOCs) (Larry A. Broussard). 8.1 Introduction. 8.2 General Considerations. 8.3 Intended Use. 8.4 Volatility of Compounds. 8.5 Sample Collection, Handling and Storage. 8.6 Headspace Gas Chromatographic Methods. 8.7 Columns and Detectors. 8.8 Identification, Quantitation and Confirmation. 8.9 Ethanol and Other Volatile Alcohols. 8.10 Inhalants and Screening for Multiple VOCs. 8.11 Interpretation. 8.12 Conclusion. References. 9. Chromatographic Techniques for Measuring Organophosphorus Pesticides (H. Wollersen and F. Musshoff). 9.1 Introduction. 9.2 Organophosphorus Pesticides (OPs). 9.3 Conclusion. References. 10. Chromatographic Analysis of Nerve Agents (Jeri D. Ropero-Miller). 10.1 Introduction. 10.2 Neuromuscular Blockers. 10.3 Paralytic Shellfish Poisoning: Saxitoxin. 10.4 Summary. References. 11. History and Pharmacology of c-Hydroxybutyric Acid (Laureen Marinetti). 11.1 Introduction. 11.2 History of Illicit Use of GHB. 11.3 Clinical Use of GHB in Humans. 11.4 History of Illicit Use of GBL and 1,4BD. 11.5 Distribution and Pharmacokinetics of GHB, GBL and 1,4BD. 11.6 GHB Interpretation Issues and Post-mortem Production. 11.7 Analysis for GHB, GBL and 1,4BD. References. 12. Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometric Detection for Element Speciation: Clinical and Toxicological Applications (Katarzyna Wrobel, Kazimierz Wrobel and Joseph A. Caruso). 12.1 Introduction. 12.2 Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometric Detection. 12.3 Analytical Applications of Clinical and Toxicological Relevance. 12.4 Conclusions and Future Trends. 12.5 Abbreviations. References. 13. Applications of Gas Chromatography-Mass Spectrometry to the Determination of Toxic Metals (Suresh K. Aggarwal, Robert L. Fitzgerald and David A. Herold). 13.1 Introduction. 13.2 Instrumentation. 13.3 Experimental Procedure. 13.4 GC-MS Studies. 13.5 Conclusions. References. Index..

Choosing the right laboratory: A review of clinical and forensic toxicology services for urine drug testing in pain management

Urine drug testing (UDT) services are provided by a variety of clinical, forensic, and reference/specialty laboratories. These UDT services differ based on the principal activity of the laboratory. Clinical laboratories provide testing primarily focused on medical care (eg, emergency care, inpatients, and outpatient clinics), whereas forensic laboratories perform toxicology tests related to postmortem and criminal investigations, and drug-free workplace programs. Some laboratories now provide UDT specifically designed for monitoring patients on chronic opioid therapy. Accreditation programs for clinical laboratories have existed for nearly half a century, and a federal certification program for drug-testing laboratories was established in the 1980s. Standards of practice for forensic toxicology services other than workplace drug testing have been established in recent years. However, no accreditation program currently exists for UDT in pain management, and this review considers several aspects of laboratory accreditation and certification relevant to toxicology services, with the intention to provide guidance to clinicians in their selection of the appropriate laboratory for UDT surveillance of their patients on opioid therapy.