Advances and challenges in the measurement of 1,25-dihydroxyvitamin D: a comprehensive review.

Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH)2D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH)2D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH)2D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH)2D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH)2D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH)2D measurement is needed to ensure accurate and reliable results across laboratories and methods.

Accuracy-Based Glomerular Filtration Rate Assessment by Plasma Iohexol Clearance in Kidney Transplant Donors.

BACKGROUND: An accurate measurement of the glomerular filtration rate (GFR) is essential for detecting renal insufficiency in living kidney donors. Iohexol is a "near-ideal" exogenous filtration marker for GFR measurements that has attracted increasing interest in clinical practice because it is non-toxic, non-radioactive, readily available, and easy to measure. In this study, we aimed to set up a laboratory test to conveniently assess the plasma clearance of iohexol in living kidney donors. METHODS: A workflow was established in the institution's infusion clinic to administer iohexol and to collect three timed blood samples from renal transplant donors. Iohexol was thereafter measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum proteins were precipitated and the supernatant containing iohexol was diluted prior to the LC-MS/MS analysis. The LC-MS/MS method was developed on a Thermo Vanquish UHPLC coupled with a TSQ Endura triple quadruple mass spectrometer with a total run time of 2.5 min. The analytical performance of the method was assessed. RESULTS: The LC-MS/MS method demonstrated a good analytical performance. To calculate the iohexol clearance rate and the GFR, automated data integration and a result calculation were accomplished by using a custom Python script. Automated result reporting was achieved using a laboratory informatics system (LIS) vendor's direct media interface. CONCLUSIONS: We developed and implemented a laboratory test to assess the plasma clearance of iohexol. A workflow was established in the hospital to reliably measure the GFR in living kidney donors, with a potential to be further expanded into other areas where an accurate GFR measurement is needed.

Overcome Isomer Interference in 1α,25-Dihydroxyvitamin D Quantitation by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: The circulating concentration of 1α,25-dihydroxyvitamin D [1α,25(OH)2D] is very low, and the presence of multiple isomers may lead to inaccurate quantitation if not separated prior to analysis. Antibody-based immunoextraction procedures are sometimes used to remove structurally related isomers of 1α,25(OH)2D prior to an LC-MS/MS analysis. However, immunoextraction increases sample preparation time and cost. In addition, some dihydroxyvitamin D metabolites are not completely removed by immunoextraction. METHOD: We developed an HPLC method using a phenyl-hexyl column to investigate interfering isomers of 1α,25(OH)2D. RESULT: Using this method, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization product of 1α,25(OH)2D was found to be present as 2 epimers, which were separated chromatographically with an area ratio of 2:1. PTAD derivatized metabolite of 25-hydroxyvitamin D3 [i.e., 4ß,25-dihydroxyvitamin D3 (4ß,25(OH)2D3)] eluted out between 6R and 6S epimers of derivatized 1α,25(OH)2D3. If not chromatographically resolved, 4ß,25(OH)2D can affect 1α,25(OH)2D quantitation. In a method comparison study, it was found that the presence of 4ß,25(OH)2D produced positive bias up to 127% on 1α,25(OH)2D3 quantitation. CONCLUSION: The LC-MS/MS method we developed without an immunoextraction procedure was able to resolve the major interference peak from 1α,25(OH)2D and achieved reliable quantitation of 1α,25(OH)2D.

False-Positive Enzymatic Alcohol Results in Perimortem Specimens.

Herein, we present 2 cases referred to the North Carolina Office of the Chief Medical Examiner (NC OCME) in which ethanol results reported by different hospital laboratories, using alcohol dehydrogenase (ADH)-based assays, were positive, whereas results of headspace gas chromatography testing performed in the NC OCME laboratory were negative. Literature reports suggest that false-positive ethanol measurements from ADH-based assays can occur when a combination of elevated lactate and lactate dehydrogenase (LD) are present in the specimen. The results were reported in perimortem specimens collected from 2 children with unrelated medical conditions. The cases and associated clinical parameters are considered based on the lactate/LD explanation for the false-positive results, to facilitate the recognition of circumstances that can produce erroneous serum ethanol results.

Failure of amoxicillin to produce false-positive urine screens for cocaine metabolite.

Amoxicillin has been causally linked in the lay and medical literature to false-positive urine drug screens for cocaine metabolites. An exhaustive search of the peer-reviewed medical literature revealed no data to support this link. We hypothesized that amoxicillin does not cause false-positive urine drug screens for cocaine metabolites. To test this hypothesis, we examined the urine of 33 subjects administered a course of amoxicillin, subjecting the specimens to four common urine screening immunoassays. Thirty-one specimens were negative for the cocaine metabolite, benzoylecgonine (BE), by all four screening methods; two were positive for BE by all four screening methods. Both positive specimens were confirmed by gas chromatography-mass spectrometry (GC-MS) for the presence of BE at > 150 ng/mL. Three specimens that screened negative, but produced absorbance values that were intermediate between negative and positive controls, were submitted for GC-MS analysis; BE was detected in all three specimens at concentrations of 54, 94, and 119 ng/mL. Twenty-eight specimens produced screening results indistinguishable from negative controls. Within the limitations of the study design, we conclude that amoxicillin is unlikely to produce false-positive urine screens for cocaine metabolites.

Rational use and interpretation of urine drug testing in chronic opioid therapy.

Urine drug testing (UDT) has become an essential feature of pain management, as physicians seek to verify adherence to prescribed opioid regimens and to detect the use of illicit or unauthorized licit drugs. Results of urine drug tests have important consequences in regard to therapeutic decisions and the trust between physician and patient. However, reliance on UDT to confirm adherence can be problematic if the results are not interpreted correctly, and evidence suggests that many physicians lack an adequate understanding of the complexities of UDT and the factors that can affect test results. These factors include metabolic conversion between drugs, genetic variations in drug metabolism, the sensitivity and specificity of the analytical method for a particular drug or metabolite, and the effects of intentional and unintentional interferants. In this review, we focus on the technical features and limitations of analytical methods used for detecting drugs or their metabolites in urine, the statistical constructs that are pertinent to ordering UDT and interpreting test results, and the application of these concepts to the clinical monitoring of patients maintained on chronic opioid therapy.

Opioid Use Disorders, Medication-Assisted Treatment, and the Role of the Laboratory.

Urine drug testing is an essential component in the evaluation and management of individuals with opioid use disorders, including those on buprenorphine or methadone maintenance therapies. Notwithstanding its centrality in adherence monitoring, studies have shown that clinicians have important knowledge deficiencies regarding the ordering and interpretation of drug tests. In this review, we discuss the scope and frequency of testing, the advantages and disadvantages of immunoassay- (presumptive) and liquid chromatograph-mass spectrometry- (definitive) based techniques, indications for definitive testing, medical necessity, and other considerations. Optimal use of urine drug testing depends on collaboration between clinicians and laboratory scientists.