Judith Stone (Presenter)
Univ. of Calif, San Diego Health System
Bio: Judy Stone is the Senior Technical Specialist in the Clinical Mass Spectrometry Laboratory, Univ. of Calif. San Diego Health Center for Advanced Laboratory Medicine. Her research interests are in mass spectrometry method development and automation for clinical toxicology, therapeutic drug monitoring, and endocrinology. She teaches a short course twice yearly on "Getting started with quantitative clinical liquid chromatography-tandem mass spectrometry" at the Mass Spectrometry Applications for the Clinical Laboratory meetings in the United States and Europe. She is Chief of the Editorial Board for the Mass Spectrometry Feature Series in Clinical Laboratory News, American Association of Clinical Chemistry.
Authorship: Stone, Judith A (1), Pesce Amadeo J (2) Fizgerald Robert L (3).
(1) UC San Diego Health Center for Advanced Laboratory Medicine (2) UC San Diego School of Medicine, Precision Diagnostics LLC (3) UC San Diego Dept. of Pathology
Sublingual buprenorphine is used for treatment of opioid abuse. Naloxone:buprenorphine coformulation is common to deter parenteral misuse. Naloxone has low sublingual bioavailability, therefore limited antagonist effect by this route, whereas intravenous naloxone is a highly effective opioid antagonist. Urine drug testing may detect diversion/misuse of BNX. Conventional wisdom is that naloxone is not detected in urine after sublingual BNX. Using LC-MSMS we quantified total urine buprenorphine, norbuprenorphine and naloxone from two patient populations prescribed BNX (A&B, nA=44,079,nB=49). We found higher than expected concentrations of naloxone. In populations A/B naloxone was detected in 91%/92% of samples with minimum-median-maximum A/B concentrations of 10/8-581/206-168,207/2,243 ng/mL. We discuss analytical, clinical and pharmacokinetic explanations for this observation.
Sublingual buprenorphine is used for treatment of opioid use disorder. Buprenorphine:Naloxone co-formulation in a 4:1 ratio is common to deter parenteral misuse (nasal or intravenous administration). Naloxone has minimal sublingual bioavailability, therefore limited antagonist effect by this route, whereas intravenous naloxone is a highly effective opioid antagonist. Urine drug testing is used to help detect diversion/misuse of BNX. Conventional wisdom is that naloxone >100 ng/mL should not be detected in urine when patients are compliant with sublingual BNX as prescribed. We validated an LC-MSMS method (B) designed to measure total urine concentrations of buprenorphine, norbuprenorphine and naloxone (BPNBX),after enzymatic hydrolysis of glucuronide metabolites, with lower limits of quantitation (LLoQ) of 0.5, 0.5 and 5 ng/mL respectively. We expected the majority of patient urine samples tested during validation would have undetectable naloxone concentrations. Instead naloxone was measurable in 92% of samples (sample set B) with concentrations between 8-2,243 ng/mL. We initiated an investigation to determine whether our method or our expectations were in error.
To investigate this unexpected outcome, we compared total BPNBX results for 20 samples with a validated LC-MSMS method (A) from a pain management reference laboratory.
Both methods used genetically modified beta-glucuronidase enzyme from Integrated Micro-Chromatography Systems, LLC (IMCS-Columbia, SC). Method A/B hydrolysis conditions were 30 min at 60/65 °C with 1,500/1,000 enzyme units/well. The efficacy of glucuronide hydrolysis was evaluated for method B by adding equimolar free and glucuronide BPNBX standards near the upper limit of quantitation to separate aliquots of ten patient urines (BPNBX negative samples). Samples were processed in duplicate with method B and hydrolysis recovery was calculated as mean BPNBX peak areas in each glucuronide analyte spiked sample divided by mean BPNBX peak areas in the matching free analyte spiked sample. Possible interfering substances for method B were tested by addition of 65 abused, prescription and over-the-counter drugs/metabolites to aliquots of drug-free urine at concentrations from 5,000 to 50,000 ng/mL. The spiked samples were processed with method B.
Method A used a dilute and shoot sample preparation, method B used an AC extraction plate™ (Tecan Schweiz AG-Männedorf, Switzerland) method. Samples with naloxone concentrations >1,000 ng/mL were analyzed on dilution (x10) with method B and undiluted with method A. The methods had minor differences in LC column, mobile phases, and gradient. The same naloxone quantifier MRM (328/212 m/z) and naloxone-d5 internal standard were used with different qualifier MRMs. MSMS instruments were A-SCIEX 6500 and B-Waters XEVO TQS.
Frequency distributions for analyte concentrations and ratios were analyzed for two data sets: sample set A, n=44,079,from the pain management reference laboratory and sample set B, n=49, collected at 1.an academic medical center and 2.the core laboratory for a regional health care system.
Urine collection early or late in the post-dose interval:
Several authors have proposed that the urine norbuprenorphine:buprenorphine ratio (NB:BP) may be an indicator of the time since last dose (George 2004, Bottcher 2005, Kronstrand 2008). Kronstrand found for single sublingual BNX dosing that a NB:BP ratio <0.5 was an indicator of very recent use, a ratio of 1 indicated use within 7-10 hrs and a ratio of 3 indicated use 1 day before sampling. We compared naloxone concentrations in a subset of A samples with NB:BP ratios 0.1-0.9 (early collection) and a second subset with NB:BP ratios greater than 4.0 (late collection).
Knowledge base for naloxone pharmacokinetics:
We reviewed publications (n=23) from 1997-2016 identified through PubMed and Google searches using search terms of urine, naloxone, concentrations, pharmacokinetics, and buprenorphine.
Analytical method validation:
Samples with naloxone concentrations between 10-1,000 ng/mL (n=15) had good agreement between methods A and B. Passing-Bablok regression statistics (95% confidence limits) were slope = 1.033 (0.937 to 1.114), intercept = -1.3 (-27.5 to 11.5) and SMAD = 20.8. Biases between the two methods for all samples were within +/-15%. There was a positive B to A bias for samples with naloxone concentrations >1,000, with Deming regression slope of 1.504 and mean bias of 7.3%, probably related to dilution before analysis of these samples with method B but not with method A.
Mean recoveries of free analytes for the glucuronide hydrolysis recovery experiment with method B were naloxone 93%, norbuprenorphine 115%, buprenorphine 115%,. None of the 65 drug/metabolite compounds tested caused an interference with naloxone.
Concentrations, ratios, and frequency distributions:
Total naloxone was detected above the lower limit of quantitation in Set A/Set B (LLoQs 10 ng/mL/5 ng/mL) in 91%/92% of all samples. Minimum-median-maximum A/B total naloxone concentrations were 10/8-581/206-168,207/2,243 ng/mL.
Within the subset of samples with detectable naloxone, we defined a further subset of samples (Set A-Admin/Set B-Admin) that were likely to represent true BNX administration and unlikely to have been adulterated by illicit addition of BNX tablet or film directly to the urine container. The criteria for true administration were total buprenorphine concentration less than 1,000 ng/mL, total norbuprenorphine metabolite concentration greater than 5 ng/mL and a total norbuprenorphine:total buprenorphine ratio greater than 0.1 (Hull 2008). Of samples with detectable naloxone, the majority, 93% and 98% (Set-A/Set B) - were consistent with our criteria for true administration rather than adulteration. In these Admin subsets, 74% and 71% (Set A/Set B) of samples had total naloxone concentrations greater than 100 ng/mL.
In the Set A-Admin samples-we compared the distribution of total naloxone concentrations less than 500 versus greater than or equal to 500 ng/mL in the early and late collection subsets (NB:BP ratio 0.1-0.9 early and greater than 4.0 late). Early collection samples were predominantly greater than or equal to 500 (40% less than 500 and 60% greater than or equal to 500) and late collection samples were more often less than 500 (76% less than 500 and 24% greater than or equal to 500).
We found one reference that described detection of total naloxone (concentration range 5-1,700 ng/mL) in all urine samples collected from 40 patients on BNX therapy (Heikman 2014). One reference described naloxone as not present in a random subset of 87 samples presumed to be from BNX compliant patients (free buprenorphine <20 ng/mL and free norbuprenorphine >20 ng/mL), but clearly specified that only free naloxone was analyzed (McMillin 2012). We found 4 publications describing naloxone (undefined as to free or total) as undetectable in urine after sublingual BNX administration or stating that naloxone was not absorbed or very poorly absorbed sublingually (Harris 2000, Mendelson 2004, Elkader 2005, Hull 2008). The remainder of the references reviewed either measured naloxone only in plasma and stated that low or undetectable concentrations were a consequence of low sublingual bioavailability rather than low sublingual absorption or did not measure naloxone.
Conclusions & Discussion
Possible explanations for our detection of total urine naloxone greater than 100 ng/mL in the majority of samples from patients taking BNX include analytical error, a high frequency of urine sample adulteration with a BNX tablet/film, high frequency of intranasal/intravenous BNX use, or the wrong expectation that total naloxone urine concentrations should be less than 100 ng/mL.
Based on the method comparison and validation studies it seems unlikely that analytical error in our methods caused consistent over-estimation of naloxone concentrations to the extent we observed.
We found only 2-3% of samples suggestive of adulteration, which cannot explain the high frequency (91-92% of all samples) in which naloxone was detected in our sample sets.
Naloxone has extensive, rapid first-pass metabolism to naloxone-glucuronide and other metabolites after sublingual or oral absorption, yielding low free naloxone concentrations in blood and urine. Comparatively, the bioavailability and therefore urine concentrations of free naloxone are higher following intramuscular, intranasal or intravenous administration (Fan 2009, Wermeling 2013). For our sample sets, we cannot rule out higher total naloxone concentrations than expected as a consequence of frequent intranasal/intravenous BNX use (Jones 2014) but one study in Australia estimated the frequency of BNX film/tablet injection for patients in treatment as only 3-9% (Lofwal 2014).
A wrong expectation that total naloxone concentrations will be less than 100 ng/mL in urine from patients treated with sublingual BNX appears to be the most likely explanation. A careful study of selected literature is consistent with the interpretation that free naloxone concentrations are low in urine after sublingual BNX administration, but total naloxone (free + naloxone-glucuronide measured after hydrolysis to free naloxone) may be easily detected using methods with an LLoQ of 1-10 ng/mL. However, a quick review without consideration as to the method used for naloxone analysis (free versus total), as might be performed after an urgent query from a clinician, could easily lead to the false conclusion that naloxone greater than 100 ng/mL is never detectable in urine samples from patients treated with sublingual BNX except from adulteration,.
Our explanation for measuring higher than expected urine total naloxone concentrations is that naloxone is very rapidly and extensively metabolized after sublingual absorption to naloxone glucuronide, which is renally eliminated in relatively high concentrations within the first 1-2 hours after once-daily sublingual administration. The distribution of total naloxone concentrations in our sample sets, higher in samples likely to have been collected early rather than late in the post-dose interval, supports this interpretation. We conclude that detection of total urine naloxone greater than 100 ng/mL is common for patients treated with sublingual BNX. We encourage diagnostic and research laboratories measuring urine naloxone to clearly distinguish between measurement of free naloxone, naloxone-glucuronide, and total naloxone in their reporting to avoid mis-interpretation.
References & Acknowledgements:
We thank Judy Chang, Scientific Director, The Kaiser Permanente Medical Group Northern California Regional Laboratory, for sharing de-identified remnant urine samples and results for buprenorphine and norbuprenorphine. We thank Agnieszka Sitarska, Product Manager, Tecan Schweiz AG (Männedorf, Switzerland) for collaboration on the AC plate BPNBX LC-MSMS method.
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