Assessment of mass spectrometry-based pain management testing in clinical laboratories across North America.

BACKGROUND: Urine drug testing (UDT) is a useful tool in monitoring compliance to prescribed medication and can also help identify behaviors of drug misuse, abuse, and diversion. Mass spectrometry (MS)-based screening is recommended as the first-line of UDT for pain management patients; however, this testing comes with an inherent lack of standardization in methodologies and various analytical challenges. The objective of this study was to assess the current state of UDT for pain management in a cross-section of clinical laboratories in North America. MATERIALS AND METHODS: A total of 10 blinded urine samples were sent to 6 laboratories across the United States and Canada. Urine samples containing drugs and/or metabolites of interest were included to represent different clinical scenarios commonly seen in pain management settings. Assessment was based on the ability of the laboratories to correctly identify drugs and provide a meaningful interpretation of the findings (when offered by the performing laboratory). RESULTS: Across the laboratories involved in the study, 85% of tests correctly identified and appropriately reported the drugs present in the urine samples. Similarly, 84% of samples were considered to have an accurate interpretation included in the UDT report. Out of the total number of drugs included in the samples, 11% were not offered on every test menu. CONCLUSIONS: This study revealed the lack of standardization in pain management UDT performed in a limited cross-section of clinical laboratories across North America.

Determination of urinary prostaglandin E2 as a potential biomarker of ureteral stent associated inflammation.

Ureteral stents are the most widely used surgical implant in urology. However, they may cause adverse effects to patients, including pain, discomfort, and inflammation. In this work, the inflammatory effect of stent placement and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed. Furthermore, a capillary electrophoresis mass spectrometry (CE-MS) based approach was subsequently developed to quantify urinary prostaglandin E2 (PGE2), a COX-2 metabolite known to contribute to inflammatory renal diseases, to further interrogate the role of this pathway. Urine samples were cleaned and preconcentrated by solid-phase extraction (SPE), and an on-line sample stacking method was used for the enrichment of analytes. The accuracy, precision, and specificity of this method were validated. Standard addition methods were performed to assess the reliability of using deuterated internal standards (IS) in compensating the remaining matrix effect after SPE as well as the detector fluctuation. Through the analysis of 32 pig urine samples, a statistically significant increase of PGE2 was observed in the stented group compared to the unstented (P = 0.01) and the recovered (P = 0.004) groups. This work determined that stent placement may contribute to COX-2-dependent inflammation and developed a reliable CE-MS based methodology to quantify PGE2 in stented individuals that may further understand the biology of stent-associated inflammation and inform urologic patient management.

Effect of Water Avoidance Stress on serum and urinary NGF levels in rats: diagnostic and therapeutic implications for BPS/IC patients.

Nerve growth factor (NGF) is thought to play a key role in chronic pain felt by bladder pain syndrome/interstitial cystitis (BPS/IC) patients by activating its high affinity receptor tropomyosin-related kinase subtype A (Trk A). Whether this pathway is also involved in the aggravation of pain sensation during stress events was here investigated. The levels of plasmatic NGF were increased in rats submitted to Water Avoidance Stress test (WAS), compared to controls. The administration of the alpha1A adrenoceptors blocker silodosin prevented the increase of plasmatic NGF. Urinary NGF levels were also moderately increased in animals submitted to WAS. WAS increased pain behaviour score, lowered abdominal mechanical pain threshold and increase voiding bladder reflex activity. These changes were prevented by the administration of TrkA antagonist GW441756. These findings prompt the use of plasmatic NGF as diagnosis tool for chronic visceral painful conditions and opens therapeutic opportunities for TrkA receptors antagonist/NGF sequestration.

Consumption of Movantik™ (Naloxegol) results in detection of naloxone in the patient's urine evaluated by confirmatory urine drug testing.

BACKGROUND: Many patients on chronic opioid therapy suffer from constipation, one of the most common side effect of opioids. Movantik™ (naloxegol) is an opioid antagonist that is recently introduced in the market to treat opioid-induced constipation and contains naloxegol as the active ingredient. Naloxegol is a pegylated (polyethylene glycol-modified) derivative of α-naloxol. Detection of naloxone in the patients urine after consumption of naloxegol was not reported by the manufacturer and may mislead the prescribing clinicians. This study was conducted to investigate the presence of naloxone in the urine of patients that consume movnatik in pain management clinics. METHODS: The presence of naloxone and naloxol in the urine of 45 patients that consumed naloxegol and 25 patients that consumed suboxone™ were investigated using a liquid chromatography mass spectrometry (LCMS) method. The urinary concentration of naloxone, naloxol, and their glucuronide conjugates were evaluated in five volunteers that took one pill of naloxegol for one day and one volunteer who took the pill for three days. RESULTS: Naloxone was detected in the urine of 45 individuals that were prescribed naloxegol. Urinary concentration of naloxone showed a distribution with a mean of 25 ±â€¯18 ng/ml. Consumption of one pill of 25 mg naloxegol resulted in the detection of naloxol and naloxone in the urine of 5 volunteers 1 h after taking the pill. Evaluation of urine specimens from 25 patients that consumed suboxone™, resulted in the detection of naloxone (180 ±â€¯187 ng/ml) and naloxol (6.3 ±â€¯7.2 ng/ml). CONCLUSIONS: This study demonstrated that consumption of naloxegol leads to appearance of naloxone in the urine of patients receiving opioid therapy in pain management clinics.

Anodyne by Design; Measuring the Prevalence of Esoteric Designer Opioids in Pain Management Patients.

The recent increase in illicit opioids sold on the black market, cut into heroin and masqueraded as prescription pills prompts a significant public health concern. Most designer opioids possess unknown potencies and unknown pharmacokinetics and their unregulated, variable dosages lead to rashes of overdoses. Additionally, many of the designer opioids, especially the fentanyl analogs are significantly more potent than heroin. High-profile cases involving overdoses of U-47700 and carfentanil have been reported in the media; however, the true prevalence of these and other designer opioids is unknown. Independent LC-MS-MS screen and confirmation methods have been developed and validated to identify and quantify fentanyl, and 18 designer opioids and their metabolites; methods were then exercised on urine specimens from contract pain management clients. Assuming patients in a pain management program may have a higher probability to seek out self-medication, samples from pain management patients were investigated for designer opioids. Similarly, pain management patients identified as using heroin may be more likely to experiment with or be accidentally exposed to designer opioids, specimens screening positive for the heroin metabolite 6-acetylmorphine were specifically chosen for designer opioid screening. Within this small group of pain management and heroin-positive samples, nine designer opioids were detected at a total prevalence of 25%. When screening random pain management samples not positive for heroin, a considerably lower percentage of samples (<1%) were identified as positive for designer opioids. Furanyl fentanyl, fluorobutyryl fentanyl and acetylfentanyl were the most prevalent designer opioids detected in both test groups.

The water avoidance stress induces bladder pain due to a prolonged alpha1A adrenoceptor stimulation.

Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) remains an elusive disease with the cause for the pain unclear. BPS/IC patients present increased sympathetic activity and high levels of urinary noradrenaline. At the experimental level, it has been shown that chronic adrenergic stimulation produces pain and bladder changes through an alpha 1A adrenoceptor mediated mechanism. Water avoidance stress (WAS) in rodents reproduces signs of nociception and bladder changes seen in BPS/IC patients. In this study, we explore the possible role of alpha 1A adrenoceptor in bladder pain and morphological changes. WAS was induced in a group of female Wistar rats. A separate WAS group received 0.2 mg/kg day silodosin (WAS + S). Lower abdominal pain was determined by performing sensitivity to Von Frey filaments. Bladder reflex activity was determined by cystometry in anaesthetised animals. Urine was collected for noradrenaline quantification by HPLC. Bladders were harvested and stained with Haematoxylin-eosin (to analyse urothelial morphology and to determine the disruption of surface umbrella cells) or with Toluidine Blue 0.1% to analyse mast cell infiltration. WAS increased urinary noradrenaline level and bladder frequency and decreased mechanical pain threshold, which was reversed by silodosin. WAS induced lymphocytic and mast cells infiltration in the mucosa and mild urothelial disruption, which was absent in WAS + S group. Alpha 1A adrenoceptor stimulation has an important role in the appearance of bladder pain in rats. Since BPS/IC patients present high levels of noradrenaline, alpha 1A stimulation may be an additional trigger for bladder dysfunction presented by these patients. Further studies will determine the clinical relevance of this finding in the treatment of BPS/IC patients.

Toxicology in Pain Management.

Toxicology testing in pain management has become the standard of care. The Center for Disease Control and Prevention published guidelines including urine drug testing prior to initiating opioid therapy and for monitoring prescription and illicit drugs. Physicians must know indications for toxicology testing, and the frequency based upon risk stratification. This includes personal and family history of alcohol or substance related disorders, mental illness and smoking. Knowledge of opioid metabolism and various matrices to test are described. Additional knowledge of presumptive vs definitive testing along with algorithms and medical necessity with evidence-based clinical standards are discussed.

Urine Creatinine Concentrations in Drug Monitoring Participants and Hospitalized Patients.

Urine drug testing is commonly performed in both clinical and forensic arenas for screening, monitoring and compliance purposes. We sought to determine if urine creatinine concentrations in monitoring program participants were significantly different from hospital in-patients and out-patients undergoing urine drug testing. We retrospectively reviewed urine creatinine submitted in June through December 2015 for all specimens undergoing urine drug testing. The 20,479 creatinine results were categorized as hospitalized patients (H) and monitoring/compliance groups for pain management (P), legal (L) or recovery (R). Median creatinine concentrations (interquartile range, mg/dL) were significantly different (P < 0.001) between groups: H 126 (122-136); P 138 (137-143); L 147 (144-154); R 95 (92-97). In the two groups subject to on-demand sampling time pressures, median creatinine concentrations were significantly lower in the R vs. L group (P<0.001). In conclusion, recovery (R) participants have more dilute specimens, reflected by significantly lower creatinine concentration and may indicate participants' attempts to tamper with their drug test results through dilution means.

Urinary cytokines/chemokines pattern in patients with painful bone metastases undergoing external beam radiotherapy experiencing pain flare.

BACKGROUND: External beam radiotherapy (EBRT) is a mainstay for treatment of painful bone metastases. Transient worsening of pain ("pain flare") occurs in 40% of patients. We investigated the pathophysiology of pain flare through assessment of changes in urinary cytokines/chemokines in patients receiving EBRT for painful bone metastases. METHODS: Urine samples were collected from patients receiving a single 8 Gy fraction for painful bone metastases preparation, day 1 or 2 and on an additional day between days 3 to 5 post radiation. Patients completed a standardized pain and analgesic use diary daily for 10 days following radiation. Patients were deemed to have pain flare if they had a two-point increase from baseline worst pain on 0-10 scale and no decrease in analgesic intake or a 25% increase in analgesic intake with no decrease in worst pain. The Millipore Milliplex 42-Plex Cyto-kine/Chemokine Kit™ was used to measure urinary levels of a panel of cytokines/chemokines. RESULTS: Forty-six patients consented to the study of which 28 were evaluable (complete urine and diary data), and 83/84 urine samples were available for analysis. Pain flare was experienced by 11 patients (39%). The following cytokines/chemokines were detectable in at least 50% of the patients: EGF, fractalkine, GRO, IL-4, IL-8, interferon gamma induced protein 10 (IP-10), MCP-1, macrophage derived chemokine (MDC), PDGF-AA, sIL-2Ra, TGF-Alpha, VEGF. Comparing patients with or without pain flare EGF, fractalkine, GRO, IL-8, IP-10, MCP-1, MDC, sIL-2Ra, and TGF-alpha increased following radiation in both groups. Patients with pain flare have significant lower levels on IL-8, IP-10, and MDC over time. No specific time trend was noticed. CONCLUSIONS: Patients who experience pain flare appear to have a different pattern in urinary cytokine/chemokine levels than patients without pain flare. A larger study is required to confirm the possible role of cytokines/chemokines in predisposition to and/or the cause of pain flare following radiation to painful bone metastases.

Profiles of Urine Drug Test in Clinical Pain Patients vs Pain Research Study Subjects.

OBJECTIVE: To examine similarities and differences in urine drug test (UDT) results in clinical pain patients and pain subjects participating in pain research studies. DESIGN: An observational study with retrospective chart review and data analysis. METHODS: We analyzed 1,874 UDT results obtained from 1) clinical pain patients (Clinical Group; n = 1,529) and 2) pain subjects consented to participate in pain research studies (Research Group; n = 345). Since several medications such as opioids used in pain management are drugs of abuse (DOA) and can result in a positive UDT, we specifically identified those cases of positive UDT due to nonprescribed DOA and designated these cases as positive UDT with DOA (PUD). RESULTS: We found that 1) there was a higher rate of PUD in clinical pain patients (41.3%) than in pain research study subjects (14.8%); 2) although subjects in the Research Group were informed ahead of time that UDT will be conducted as a screening test, a substantial number (14.8%) of pain research study subjects still showed PUD; 3) there were different types of DOA between clinical pain patients (cannabinoids as the top DOA) and research study subjects (cocaine as the top DOA); and 4) a common factor associated with PUD was opioid therapy in both Clinical Group and Research Group. CONCLUSION: These results support previous findings that PUD is a common finding in clinical pain patients, particularly in those prescribed opioid therapy, and we suggest that UDT be used as routine screening testing in pain research studies.

Urinary N telopeptide levels in predicting the anti-nociceptive responses of zoledronic acid and paclitaxel in a rat model of bone metastases.

The present study investigated the hypothesis that urinary levels of N telopeptide (NTx) can be used to predict the anti­nociceptive responses of zoledronic acid and paclitaxel on bone metastases in a rat model. Rats were implanted with intra­femur Walker 256 carcinoma cells or control solution, and were treated with either normal saline, zoledronic acid or paclitaxel on the 10th day following surgery. Mechanical allodynia was recorded and the urine collagen­crosslinked NTx values were measured prior to, and 7, 14 and 21 days following the injections. Bone sections and osteoclasts were stained on the 14th day (4 days post­injection). Furthermore, the mRNA and protein expression levels of c­fos in the spinal cord and acid­sensing ion channel 3 (ASIC3) in the dorsal root ganglion (DRG) were analyzed. The mechanical allodynia of rats was attenuated from day 14 in the zoledronic acid group and from day 21 in the paclitaxel group. A positive correlation was observed between the anti­nociceptive responses of zoledronic acid and paclitaxel, and the urinary levels of NTx (r=0.619; P<0.001). The mRNA levels of c­fos in the spinal cord and ASIC3 in the DRG in the zoledronic acid group were reduced 14 and 21 days after inoculation, and this reduction was observed in the paclitaxel group 21 days after inoculation. Low dose paclitaxel was observed to have a weaker anti­nociceptive effect on bone cancer pain, with a later­onset, compared with zoledronic acid. The results suggested that urinary levels of NTx may predict the anti­nociceptive responses of zoledronic acid and paclitaxel in a rat model of bone metastases.

Benzodiazepine in a urine specimen without drug metabolites.

PATIENT: 41-year-old Hispanic woman. Chief Complaint: Pain in the neck, joints, and shoulders that started in August 2013. History of Present Illness: The patient has a history of psychiatric illness. Her primary care physician from an outside facility had prescribed alprazolam (Xanax) to treat her depression. The patient reported that in 2011 she experienced pain in the right side of her back and was diagnosed with sciatica. In addition, she was diagnosed with systemic lupus erythematosus (SLE) based on a positive finding for antinuclear antibodies (ANA) and double-stranded DNA (ds-DNA). She had not consulted a rheumatologist before this visit, nor had she received any medication for these conditions. Her recent history of symptoms included photosensitivity, painful aphthous ulcers, transient rashes, joint pain, myalgias, and bruising. The results of the most recent evaluation of her SLE serology revealed negative ANA and ds-DNA results. The current medications that the patient has been prescribed at Santa Clara Valley Medical Center include lamotrigine (Lamictal), sertraline (Zoloft), and gabapentin (Neurontin). In April 2014, the patient requested additional pain medication, such as hydrocodone/acetaminophen (Norco), during her physician visit. Consequently, as part of her pain management therapy agreement to ensure compliance,(1) she was subjected to a urine toxicology drug screening. At this time, her urine specimen, from an unwitnessed collection, tested positive for benzodiazepines using the Syva EMIT immunoassay (Siemens AG, Munich, Germany) at the cutoff of 200 ng/mL. However, results of a confirmatory test conducted via gas chromatography-mass spectrometry (GC/MS) did not identify any benzodiazepine metabolites in her urine but instead revealed the presence of a parent drug, alprazolam. Further, the same specimen tested positive for metronidazole. A visual inspection of the specimen revealed crystals on the bottom of the cup. In December 2013, this patient had tested positive for α-hydroxyalprazolam (an alprazolam metabolite; limit of detection, 10 ng/mL) and methamphetamine. At that time, she explained her positive urine-drug-test result by stating that "somebody had put something in my drink." Family history: Her father has been diagnosed with gout, knee osteoarthritis, and enlarged heart. Her brother has been diagnosed with clinical depression. Social history: Divorced; 3 children in the custody of their father; reported having smoked 0.5 packs of cigarettes per day for 20 years; denied any alcohol intake or illicit drug use.

Analysis of codeine positivity in urine of pain management patients.

The opioids codeine and morphine have legitimate uses in managing chronic pain conditions, but they are frequently abused. Patients prescribed opioids submit urine samples for medication compliance monitoring, and the interpretation of the results is complex. The purpose of this study was to evaluate the percentage of codeine- and morphine-positive urine drug tests that result from morphine use only, with the positive codeine result arising from low levels of codeine present in pharmaceutical formulations of morphine. This study included 80 urine samples which tested positive for codeine and morphine after pre-analytical hydrolysis and analysis by gas chromatography-mass spectrometry. Quantitative results were correlated with patient prescription information and immunoassay results to classify patients into one of four categories: heroin users (50%), codeine users (34%), codeine and morphine users (5%), and morphine users (11%). The percentage of codeine-positive resulting from morphine use was higher than previous estimates. Urine from patients prescribed morphine only was found to contain codeine at <1% of the morphine concentration, a ratio that was also observed in patients who used heroin. Careful analysis of urine drug testing results, including assessing the ratio of codeine to morphine (C/M), can help providers determine if patients are compliant with their pain management regimens.

The clinical impact of a false-positive urine cocaine screening result on a patient's pain management.

BACKGROUND: The urine of a patient admitted for chest and epigastric pain tested positive for cocaine using an immunoassay-based drug screening method (positive/negative cutoff concentration 150 ng/mL). Despite the patient's denial of recent cocaine use, this positive cocaine screening result in conjunction with a remote history of drug misuse impacted the patient's recommended pain therapy. Specifically, these factors prompted the clinical team to question the appropriateness of opioids and other potentially addictive therapeutics during the treatment of cancer pain from previously undetected advanced pancreatic carcinoma. OBJECTIVE: After pain management and clinical pathology consultation, it was decided that the positive cocaine screening result should be confirmed by gas chromatography-mass spectrometry (GC-MS) testing. RESULTS: This more sensitive and specific analytical technique revealed that both cocaine and its primary metabolite benzoylecgonine were undetectable (i.e., less than the assay detection limit of 50 ng/mL), thus indicating that the positive urine screening result was falsely positive. With this confirmation, the pain management service team was reassured in offering intrathecal pump (ITP) therapy for pain control. ITP implantation was well tolerated, and the patient eventually achieved excellent pain relief. However, ITP therapy most likely would not have been utilized without the GC-MS confirmation testing unless alternative options failed and extensive vigilant monitoring was initiated. CONCLUSION: As exemplified in this case, confirmatory drug testing should be performed on specimens with unexpected immunoassay-based drug screening results. To our knowledge, this is the first report of a false-positive urine cocaine screening result and its impact on patient management.

Evaluation of concomitant methylphenidate and opioid use in patients with pain.

Methylphenidate is a central nervous system simulant that is used for management of opioid-induced sedation. Sparse data exist regarding use patterns of methylphenidate and opioids in patients with pain. This retrospective data analysis evaluated concomitant methylphenidate and opioid use from physician-reported medication lists and in urine specimens of patients with pain. All specimens were analyzed and quantified with LC-MS-MS. Concomitant methylphenidate and opioid use (e.g., sample population) were compared with a baseline population of patients taking opioids. There were 3,326 patients with physician-reported use of methylphenidate. Of these, 1,089 patients were tested for the presence of methylphenidate in urine. Methylphenidate was positive in urine for 551 patients (detection rate of 50.6%). Ritalinic acid was positive in 776 patients (detection rate of 71.3%). The current study observed differences in the use pattern of methylphenidate based on opioid type. Physician-reported use revealed methadone had the highest percent difference between the sample and baseline populations (77%, P ≤ 0.05). Fentanyl, morphine and hydromorphone also had higher percent differences of 19.6, 25.3 and 32.3%, respectively. Further studies need to examine the apparent discrepancies between the physician-reported medication lists and urine drug testing of concomitant methylphenidate and opioid use in patients with pain.

Endocrine biomarkers and symptom clusters during the menopausal transition and early postmenopause: observations from the Seattle Midlife Women's Health Study.

OBJECTIVE: During the menopausal transition and early postmenopause, participants in the Seattle Midlife Women's Health Study were likely to belong to one of three symptom severity classes: severe hot flashes with moderate sleep, mood, cognitive, and pain symptoms (high-severity hot flash); moderate levels of all but hot flashes (moderate severity); and low levels of all (low severity). We tested models of the differential effects of hypothalamic-pituitary-ovarian (HPO) axis, hypothalamic-pituitary-adrenal (HPA) axis, and autonomic nervous system (ANS) biomarkers on the three symptom severity classes. METHODS: The Seattle Midlife Women's Health Study participants recorded symptoms monthly in diaries and provided overnight urine samples several times per year that were analyzed for estrone, follicle-stimulating hormone (FSH), cortisol, testosterone, epinephrine, and norepinephrine. Multilevel latent class analysis with multinomial regression was used to determine the effects of HPO axis, HPA axis, and ANS biomarkers on symptom severity class membership. RESULTS: Having lower estrogen and higher FSH levels was significantly associated with belonging to the high-severity hot flash class versus the low-severity class. Having lower epinephrine and higher norepinephrine levels increased the likelihood of belonging to the high-severity hot flash class versus the low-severity class. Having lower epinephrine levels was significantly associated with belonging to the moderate-severity class versus the low-severity class. Cortisol and testosterone were unrelated to symptom severity class membership. CONCLUSIONS: The association of HPO axis biomarkers (estrogen and FSH) with the high-severity hot flash class is anticipated based on prior hot flash research, and the associations of HPA axis biomarkers are as expected based on earlier laboratory studies. The association of lower epinephrine levels with the moderate-severity class suggests that these symptoms may be mediated by the ANS.

Urinary hydrocodone and metabolite distributions in pain patients.

Hydrocodone combined with acetaminophen is commonly used for moderate pain. Hydrocodone is metabolized by cytochrome P450 (CYP) 2D6 into hydromorphone and by CYP3A4 into norhydrocodone. This was a retrospective study evaluating hydrocodone, hydromorphone and norhydrocodone distributions in urine. Urine specimens (n = 76,924) were obtained from patients on chronic opioid therapy during their first or single visit and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The patients were at least 16 years of age and had documented hydrocodone use via a medication list. There were 48,710 specimens that were positive for all three analytes. Mean hydrocodone, hydromorphone and norhydrocodone mole fractions (95% confidence interval) were 0.39 (0.38-0.39), 0.12 (0.11-0.12) and 0.49 (0.48-0.49), respectively. Hydromorphone fractions were lower in women compared with men (0.11 versus 0.13; P < 0.0001). Hydrocodone mole fractions were higher in the 65-year and older age group compared with the 16- to 39-year age group (0.4 versus 0.36; P ≤ 0.005). Concurrent use of a CYP2D6 and/or CYP3A4 inhibitor altered hydromorphone and norhydrocodone mole fractions, compared with the control group. Patient factors affect hydrocodone and metabolite mole fractions and suggest increased awareness of their contribution when attempting to interpret urine drug testing results.