Bupivacaine Metabolite Can Interfere with Norfentanyl Measurement by LC-MS/MS.

BACKGROUND: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard for the measurement of fentanyl and norfentanyl (NF) in urine and is favored over immunoassays due to its superior specificity. NF is the principal metabolite of fentanyl found in the urine and is typically present in higher abundance than fentanyl. Thus, the sensitivity and specificity of LC-MS/MS relies largely on the ability to identify and quantitate NF. METHODS: We analyzed urine specimens from women who had received bupivacaine and fentanyl for epidural analgesia during labor. We analyzed the contents of the epidural bag itself and purified bupivacaine metabolite N-desbutyl bupivacaine [or N-(2,6-dimethylphenyl)piperidine-2-carboxamide (NDB)] by LC-MS/MS. RESULTS: NDB interferes with the LC-MS/MS assay for NF. NDB passes through the Q1 mass selection filter because it is isobaric with the NF precursor ion (233 m/z). Further, it shares product ions with NF (84 m/z and 150 m/z), used as quantifier and qualifier ions, respectively, in our urine NF detection method. Baseline resolution of NDB and NF using these quantifier and qualifier ions could not be achieved. A unique product ion of NF (177 m/z) was useful for distinguishing NDB from NF. CONCLUSION: Bupivacaine is a commonly used drug. Recognition of this interference by laboratories is critical for preventing the misidentification of NF, which can have profound effects on patient care.

Comparison of Oral Fluid and Urine for Detection of Fentanyl Use Using Liquid Chromatography with Tandem Mass Spectrometry.

BACKGROUND: We compared oral fluid (OF) and urine (UR) for detection of fentanyl (FEN) use in addiction medicine-psychiatry (AMP) clinics. METHODS: We measured FEN and norfentanyl (NRFEN) in UR with a limit of detection (LOD) of 2.0 µg/L and FEN in OF with an LOD of 0.5 µg/L by LC-MS/MS in 311 paired samples and compared the 2 matrices when higher OF and UR LODs were used. RESULTS: Urine (UR) detected more FEN use than OF using a LOD of 2.0 µg/L and 0.5 µg/L, respectively. FEN and/or NRFEN were detected in 44 and 59 UR specimens, respectively, and FEN in 46 OF specimens (43 OF+UR+, 3 OF+UR-, 16 OF-UR+, and 249 OF-UR-). In UR there were no instances with FEN positive and NORFEN negative. UR creatinine was <20 mg/dL in the 3 OF+UR- specimen pairs. The median OF/UR analyte concentration ratios in positive sample pairs were 0.23 for OF FEN/UR FEN and 0.02 for OF FEN/UR NRFEN. CONCLUSIONS: We demonstrate that UR detects more FEN use than OF in an AMP setting when UR FEN and UR NORFEN LODs of 2.0 µg/L are used. OF is less sensitive than UR in detecting FEN use, but is still valuable for cases with low UR creatinine and/or suspected adulteration or substitution of UR. The UR vs OF comparison statistics are greatly impacted by even minimal adjustments of the LOD.

Urinary NGAL as a Diagnostic and Prognostic Marker for Acute Kidney Injury in Cirrhosis: A Prospective Study.

INTRODUCTION: Urinary neutrophil gelatinase-associated lipocalin (NGAL) has shown promise in differentiating acute tubular necrosis (ATN) from other types of acute kidney injuries (AKIs) in cirrhosis, particularly hepatorenal syndrome (HRS). However, NGAL is not currently available in clinical practice in North America. METHODS: Urinary NGAL was measured in a prospective cohort of 213 US hospitalized patients with decompensated cirrhosis (161 with AKI and 52 reference patients without AKI). NGAL was assessed for its ability to discriminate ATN from non-ATN AKI and to predict 90-day outcomes. RESULTS: Among patients with AKI, 57 (35%) had prerenal AKI, 55 (34%) had HRS, and 49 (30%) had ATN, with a median serum creatinine of 2.0 (interquartile range 1.5, 3.0) mg/dL at enrollment. At an optimal cutpoint of 244 µg/g creatinine, NGAL distinguished ATN (344 [132, 1,429] µg/g creatinine) from prerenal AKI (45 [0, 154] µg/g) or HRS (110 [50, 393] µg/g; P < 0.001), with a C statistic of 0.762 (95% confidence interval 0.682, 0.842). By 90 days, 71 of 213 patients (33%) died. Higher median NGAL was associated with death (159 [50, 865] vs 58 [0, 191] µg/g; P < 0.001). In adjusted and unadjusted analysis, NGAL significantly predicted 90-day transplant-free survival (P < 0.05 for all Cox models) and outperformed Model for End-Stage Liver Disease score by C statistic (0.697 vs 0.686; P = 0.04), net reclassification index (37%; P = 0.008), and integrated discrimination increment (2.7%; P = 0.02). DISCUSSION: NGAL differentiates the type of AKI in cirrhosis and may improve prediction of mortality; therefore, it holds potential to affect management of AKI in cirrhosis.

Comparison of Oral Fluid and Urine for Detection of Cocaine Abuse Using Liquid Chromatography with Tandem Mass Spectrometry.

BACKGROUND: Requests for urine (UR) and oral fluid (OF) drug testing at our institutions are increasing. However, few studies have assessed the accuracy of each matrix using paired specimens and LC-MS/MS. We compared OF and UR for detection of cocaine (COC) abuse in addiction medicine-psychiatry (AMP) clinics. METHODS: We measured COC and benzoylecgonine (BZE) in OF (limit of detection (LOD) 2.0 µg/L) and BZE in UR (LOD 5 µg/L) by LC-MS/MS in 258 paired samples, and compared the two matrices when higher UR cutoffs of 25, 50, and 150 µg/L were employed. RESULTS: UR detected more COC abuse than OF at the LOD (5 µg/L). BZE was detected in 63 UR specimens and COC and/or BZE in 40 OF specimens (29 OF+UR+, 11 OF+UR-, 34 OF-UR+). UR creatinine was lower in OF+UR- specimens. COC and BZE were detected in 88% (35/40) and 75% (30/40) of OF specimens, respectively. OF was equivalent to UR at detecting COC abuse using a 25 µg/L cutoff, and detected more COC abuse than UR using 50 and 150 µg/L cutoffs. The ratio of OF COC/BZE increased with decreasing UR BZE concentrations. CONCLUSIONS: We demonstrate that OF detects more COC abuse in an AMP setting when UR BZE cutoffs ≥ 50 µg/L are utilized, and that UR creatinine concentrations are significantly lower in specimens positive for COC and/or BZE in OF and negative for BZE in UR. The presence of only COC in OF and low concentrations of UR BZE likely indicates remote use of COC.

Urine is superior to oral fluid for detecting buprenorphine compliance in patients undergoing treatment for opioid addiction.

BACKGROUND: Buprenorphine (BUP) is commonly used in opioid agonist medication-assisted treatment (OA-MAT). Oral fluid (OF) is an attractive option for compliance monitoring during OA-MAT as collections are observed and evidence suggests that OF is less likely to be adulterated than urine (UR). However, the clinical and analytical performance of each matrix for monitoring BUP compliance has not been well studied. METHODS: We collected 260 paired OF and UR specimens. Concentrations of buprenorphine (BUP) and norbuprenorphine (NBUP) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in each matrix. The glucuronide metabolites and naloxone concentrations were also measured in UR by LC-MS/MS. Medications were reviewed and UR creatinine concentrations were determined. RESULTS: 147/260 specimens (57%) were positive for BUP and/or metabolites in one or both matrices. BUP and/or metabolites were more likely to be detected in UR (p < 0.001). 1 OF specimen and 12 UR specimens were considered adulterated/substituted. The majority of patients prescribed BUP were positive for BUP in UR (97%) as opposed to OF (78%). The detection of undisclosed use approximately doubled in UR. CONCLUSIONS: UR is the preferred matrix for detecting both buprenorphine compliance and undisclosed use. Clinicians should consider the ease of collection, risk of adulteration and detection of illicit drug use when deciding on an appropriate matrix. If OF testing is clinically necessary, UR should be measured in conjunction with OF at least periodically to avoid false negative BUP results.

Vortioxetine use may cause false positive immunoassay results for urine methadone.

BACKGROUND: Urine immunoassays are frequently employed for methadone screening because they are relatively inexpensive and widely available. However, immunoassays are notoriously prone to false positives. We report that the use of vortioxetine (Trintellix® in the USA and Canada, Brintellix® worldwide) could cause false positives in the Roche KIMS Methadone II Urine immunoassay (MDN2). METHODS: We performed a spiking study using a parent drug vortioxetine concentration of 7500 ng/ml. RESULTS: Urine specimens from seven patients on typical vortioxetine doses tested positive for methadone in the Roche assay but negative for methadone in a confirmatory (GC/MS) assay and two other immunoassay platforms. Because of the pharmacokinetics of vortioxetine and the high cross-reactivity of a metabolite in the MDN2 assay, routine use of the drug could cause false positives even without detectible parent drug in the urine. CONCLUSIONS: Vortioxetine is commonly prescribed for mood disorders, which have high prevalence in patients treated for opioid addiction. For that reason, it is important that clinicians are aware of this interference.

Quality Control Practices for Chemistry and Immunochemistry in a Cohort of 21 Large Academic Medical Centers.

OBJECTIVES: In the United States, minimum standards for quality control (QC) are specified in federal law under the Clinical Laboratory Improvement Amendment and its revisions. Beyond meeting this required standard, laboratories have flexibility to determine their overall QC program. METHODS: We surveyed chemistry and immunochemistry QC procedures at 21 clinical laboratories within leading academic medical centers to assess if standardized QC practices exist for chemistry and immunochemistry testing. RESULTS: We observed significant variation and unexpected similarities in practice across laboratories, including QC frequency, cutoffs, number of levels analyzed, and other features. CONCLUSIONS: This variation in practice indicates an opportunity exists to establish an evidence-based approach to QC that can be generalized across institutions.

Monitoring opioid and benzodiazepine use and abuse: Is oral fluid or urine the preferred specimen type?

BACKGROUND: Oral fluid (OF) has become an increasingly popular matrix to assess compliance in pain management and addiction settings as it reduces the likelihood of adulteration. However, drug concentrations and windows of detection are not as well studied in OF as in urine (UR). We compared the clinical utility and analytical performance of OF and UR as matrices for detecting common benzodiazepines and opioids. METHODS: OF and UR concentrations of 5 benzodiazepines and 7 opioids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 263 paired OF and UR specimens. UR creatinine was measured and prescription medications were reviewed. RESULTS: The benzodiazepines 7-aminoclonazepam, lorazepam, and oxazepam exhibited statistically higher detection rates in UR. For opioids, 6-AM was statistically more likely to be detected in OF, while hydromorphone and oxymorphone were statistically more likely to be detected in UR. Chemical properties including glucuronidation explain preferential detection in each matrix, not UR creatinine nor prescription status. CONCLUSION: We found that OF is the preferred matrix for 6-AM, while UR is preferred for 7-aminoclonazepam, lorazepam, oxazepam, hydromorphone, and oxymorphone. However, OF should be considered if the risk of adulteration is high and use and/or misuse of benzodiazepines, hydromorphone, and oxymorphone is low.

The New Substance Abuse and Mental Health Services Administration Oral Fluid Cutoffs for Cocaine and Heroin-Related Analytes Applied to an Addiction Medicine Setting: Important, Unanticipated Findings with LC-MS/MS.

BACKGROUND: We implemented oral fluid (OF) as an alternative specimen type to urine for detection of cocaine (COC) and opiate abuse in outpatient addiction medicine clinics. METHODS: We implemented a 2-µg/L limit of quantification OF LC-MS/MS assay and compiled and reviewed all findings from a 22-month collection period for COC, benzoylecgonine (BZE), codeine (COD), 6-acetylmorphine (MAM), and morphine (MOR). We also compared the results of our clinical samples at different OF cutoffs and analytes specified in the new 2015 SAMHSA OF guidelines. RESULTS: Of 3608 OF samples, COC and BZE were positive in 593 and 508, respectively. COC or BZE was positive in 662 samples. Importantly and unexpectedly, 154 samples were COC positive and BZE negative, with 125 having COC 2.0-7.9 µg/L. A simulation with the new guideline cutoffs confirmed 65% (430 of 662) of all COC- or BZE-positive data set samples. Similarly, the new guidelines confirmed 44% (263 of 603) of data set samples positive for MOR or COD. Simulation found that the new, lower MAM guideline cutoffs detected 89% of the 382 MAM-positive samples in the data set, 104 of which the new guidelines had identified as negative for MOR and COD. CONCLUSIONS: COC (not BZE) is the dominant low-concentration OF analyte in an addiction medicine setting. This information will aid OF test interpretation. It also illustrates the importance of the 2015 guideline's new immunoassay cross-reactivity requirements and the likely improvement in detection of heroin use stemming from the new, lower MAM cutoffs.

7-aminoclonazepam is superior to clonazepam for detection of clonazepam use in oral fluid by LC-MS/MS.

BACKGROUND: Clonazepam (CLON) is not only frequently prescribed in addiction management but is also commonly abused. Therefore many addiction clinics perform oral fluid (OF) testing, which unlike urine is not subject to adulteration, to monitor CLON compliance. However, CLON and other benzodiazepines can be challenging to detect in OF due to their weakly acidic nature and their presence in low concentrations. We determined the optimal technical and clinical approach for the detection of CLON use using OF. METHODS: We measured CLON and its primary metabolite 7-aminoclonazepam (7AC) by liquid chromatography-tandem mass spectrometry in OF specimens over a 2 month period. The samples were collected using the Orasure Intercept OF sample collection device. RESULTS: One hundred samples were presumptive-positive for 7AC and/or CLON. 91 (91.0%) confirmed positive for 7AC (median, range: 4.2, 0.5-316.7 ng/ml) using the ion ratio test, while only 44 of the 100 (44.0%) samples confirmed positive for CLON (median, range: 3.7, 0.5-217.2 ng/ml) using the ion ratio test. In OF the levels of 7AC were approximately 2.4-fold higher than CLON. The use of 7AC as an analyte for the detection of both CLON compliance and undisclosed use is also recommended. CONCLUSIONS: 7AC should be the analyte measured in OF for the detection of CLON use.

Improved buprenorphine immunoassay performance after urine treatment with ß-glucuronidase.

Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with ß-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods.

A new highly specific buprenorphine immunoassay for monitoring buprenorphine compliance and abuse.

Urine buprenorphine screening is utilized to assess buprenorphine compliance and to detect illicit use. Robust screening assays should be specific for buprenorphine without cross-reactivity with other opioids, which are frequently present in patients treated for opioid addiction and chronic pain. We evaluated the new Lin-Zhi urine buprenorphine enzyme immunoassay (EIA) as a potentially more specific alternative to the Microgenics cloned enzyme donor immunoassay (CEDIA) by using 149 urines originating from patients treated for chronic pain and opioid addiction. The EIA methodology offered specific detection of buprenorphine use (100%) (106/106) and provided superior overall agreement with liquid chromatography-tandem mass spectrometry, 95% (142/149) and 91% (135/149) using 5 ng/mL (EIA[5]) and 10 ng/mL (EIA[10]) cutoffs, respectively, compared to CEDIA, 79% (117/149). CEDIA generated 27 false positives, most of which were observed in patients positive for other opioids, providing an overall specificity of 75% (79/106). CEDIA also demonstrated interference from structurally unrelated drugs, chloroquine and hydroxychloroquine. CEDIA and EIA[5] yielded similar sensitivities, both detecting 96% (22/23) of positive samples from patients prescribed buprenorphine, and 88% (38/43) and 81% (35/43), respectively, of all positive samples (illicit and prescribed users). The EIA methodology provides highly specific and sensitive detection of buprenorphine use, without the potential for opioid cross-reactivity.

The trazodone metabolite meta-chlorophenylpiperazine can cause false-positive urine amphetamine immunoassay results.

Amphetamines and methamphetamines are part of an important class of drugs included in most urine drugs of abuse screening panels, and a common assay to detect these drugs is the Amphetamines II immunoassay (Roche Diagnostics). To demonstrate that meta-chlorophenylpiperazine (m-CPP), a trazodone metabolite, cross-reacts in the Amphetamines II assay, we tested reference standards of m-CPP at various concentrations (200 to 20,000 g/L). We also tested real patient urine samples containing m-CPP (detected and quantified by HPLC) with no detectable amphetamine, methamphetamine, or MDMA (demonstrated by GC MS). In both the m-CPP standards and the patient urine samples, we found a strong association between m-CPP concentration and Amphetamines II immunoreactivity (r = 0.990 for the urine samples). Further, we found that patients taking trazodone can produce urine with sufficient m-CPP to result in false-positive Amphetamines II results. At our institution, false-positive amphetamine results occur not infrequently in patients taking trazodone with at least 8 trazodone-associated false-positive results during a single 26-day period. Laboratories should remain cognizant of this interference when interpreting results of this assay.

Urinary buprenorphine concentrations in patients treated with suboxone as determined by liquid chromatography-mass spectrometry and CEDIA immunoassay.

We report on the utility of urine total buprenorphine, total norbuprenorphine, and creatinine concentrations in patients treated with Suboxone (a formulation containing buprenorphine and naloxone), used increasingly for the maintenance or detoxification of patients dependent on opiates such as heroin or oxycodone. Patients received 8-24 mg/day buprenorphine. Two-hundred sixteen urine samples from 70 patients were analyzed for both total buprenorphine and total norbuprenorphine by liquid chromatography-mass spectrometry (LC-MS-MS). Buprenorphine concentrations in all 176 samples judged to be unadulterated averaged 164 ng/mL, with a standard deviation (SD) of 198 ng/mL. Nine samples (4.2%) had metabolite-parent drug ratios < 0.02, and 33 (15.3%) had no detectable buprenorphine. The metabolite/parent drug ratio in 166 samples had a range of 0.07-23.0 (mean = 4.52; SD = 3.97). Fifteen of 96 available urine samples (16.7%) had creatinine less than 20 mg/dL. We also found sample adulteration in 7 (7.3%) available samples. Using a 5 ng/mL urine buprenorphine cutoff, the sensitivity and specificity of the Microgenics homogeneous enzyme immunoassay versus LC-MS-MS were 100% and 87.5%, respectively. The 5 ng/mL cutoff Microgenics CEDIA buprenorphine assay results agreed analytically with LC-MS-MS in 97.9% of samples.

Effect of tramadol use on three point-of-care and one instrument-based immunoassays for urine buprenorphine.

We report that use of the popular analgesic tramadol can cause false-positive urine buprenorphine results. We examined the extent of tramadol cross-reactivity in three point-of-care urine buprenorphine immunoassays (ACON, QuikStrip, and ABMC) and an instrument-based one (Cedia). We tested 29 urine samples from patients known to be taking tramadol. Ten different samples tested positive for urine buprenorphine by at least one immunoassay. Samples with positive buprenorphine screens by immunoassay were tested for total buprenorphine and total norbuprenorphine content by liquid chromatography-tandem mass spectrometry (LC-MS-MS), which confirmed that seven of the 10 positive samples were false-positives. The remaining three positive immunoassay samples had insufficient quantity for LC-MS-MS testing. No false-positives were detected with the ACON (10 ng/mL calibration cutoff) or the Cedia assay (using a 20 ng/mL calibration cutoff). All four false-positive Cedia results (using a 5 ng/mL cutoff) in this study tested negative using the ACON device. Our data suggest that tramadol use can cause false-positive urine buprenorphine immunoassays, and this effect appears to be assay-dependent. Tramadol interference with the Cedia assay is clinically relevant, especially if the 5 ng/mL calibration cutoff is used.

Interpreting tricyclic antidepressant measurements in urine in an emergency department setting: comparison of two qualitative point-of-care urine tricyclic antidepressant drug immunoassays with quantitative serum chromatographic analysis.

Patients taking tricyclic antidepressants (TCA) can experience toxicity or severe side effects. As a rapid and less technically demanding alternative to quantitative serum analysis, most laboratories offer qualitative immunoassays to assist in the evaluation of a suspected TCA overdose. However, the relationship between quantitative serum and qualitative urine levels of TCA-related compounds and their metabolites has not been comprehensively studied. Serum high-performance liquid chromatography results were compared to the qualitative urine results using the Syva Rapid Test and the Biosite Triage. Serum concentrations of amitriptyline, desipramine, doxepin, imipramine, and nortriptyline ranging from subtherapeutic to toxic triggered a positive response on both urine immunoassay devices. On the other hand, neither immunoassay uniformly detected clomipramine, even at serum levels greater than the therapeutic range. False positives due to cyclobenzaprine were more common with the Biosite assay. For virtually all positive urine TCA findings, it was not possible to determine whether the positive results corresponded to subtherapeutic, therapeutic, supratherapeutic, or toxic serum concentrations. Because urine immunoassays are the only option for many laboratories analyzing specimens for TCAs (especially in an emergency setting), clinicians must understand the limitations and interpret results in conjunction with clinical findings and/or quantitation of serum levels.

Reduced interference by phenothiazines in amphetamine drug of abuse immunoassays.

CONTEXT: Emergency department physicians frequently request urine drug screens, but many are unaware of their limitations, including the potential for false-positive results. Promethazine, a phenothiazine derivative, is used for the treatment of allergies, agitation, nausea, and vomiting. Many patients taking promethazine are subject to urine drug screens and any potential interferences are important to recognize. DESIGN: During an 11-month period, all patients presenting to the Massachusetts General Hospital emergency department who had a finding of promethazine in their serum drug screen, and who also had a urine drug screen performed, were selected for inclusion in the study. The urine drug screen results (n = 22 patients/samples) were then studied. OBJECTIVE: To determine if promethazine use can cause false-positive urine amphetamine results in widely used drug of abuse immunoassays. RESULTS: Thirty-six percent of patients taking promethazine had false-positive test results for urine amphetamines using the EMIT II Plus Monoclonal Amphetamine/Methamphetamine Immunoassay. Sixty-four percent of patients showed cross-reactivity greater than 20% higher than the blank calibrator rate. In a separate, related study, no promethazine-induced false-positive results were seen with the EMIT II Plus, Triage, and TesTcard 9 amphetamine assays, or the Triage methamphetamine assay. Reduced chlorpromazine interference was also seen with these other assays. CONCLUSIONS: False-positive urine amphetamine results can be obtained in patients taking promethazine. Promethazine metabolite(s), and not the parent compound, are the likely cause of these urine false-positive results obtained with EMIT II Plus Monoclonal Amphetamine/Methamphetamine Immunoassay. Immunoassays from different manufacturers can have very different "interference" profiles, which the pathologist and laboratory scientist must understand and relay to clinicians.