Ropinirole metabolite mimics a new psychoactive substance (4-HO-MET) in LC-MS/MS.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is often regarded as a highly reliable methodology for confirmatory testing in analytical toxicology, especially for detection of new psychoactive substances (NPS) by clinical and forensic laboratories. However, false positives still do occur and erroneous reporting can have substantial legal implications. In this study, we investigated into the mechanism behind a clinically implausible, but apparently analytically sound, finding of a NPS (4-hydroxy-N-methyl-N-ethyltryptamine; 4-HO-MET) in a urine specimen for toxicology screening by LC-MS/MS. We discovered that a ropinirole metabolite (N-despropyl-ropinirole) was the culprit of interference as it shares high structural similarities with 4-HO-MET. The chemical similarities eluded various rigorous regulatory guidelines for compound identification utilizing computer-aided spectral library matching. After careful scrutiny of the mass spectra and comparison with a reference specimen, the compound was correctly identified. Our findings emphasize the important synergy between scientists and pathologists in considering the clinical context, especially drug history, in clinical and forensic toxicology analysis on biological specimens. Mass spectra should be reviewed for relative ion ratios in case of doubt. Understanding drug metabolism is essential for troubleshooting and result interpretation.

Urine organic acid as the first clue towards aromatic L-amino acid decarboxylase (AADC) deficiency in a high prevalence area.

BACKGROUND: Aromatic L-amino acid decarboxylase deficiency is a rare neurometabolic disease due to impaired decarboxylation of neurotransmitter precursors to its active form. CASE: We retrospectively reviewed 8 cases from 2008 to 2019 with cerebrospinal fluid neurotransmitter analysis performed at our centre. All cases had an elevated urine vanillactic acid and, in most cases, with N-acetylvanilalanine detected. Cerebrospinal fluid analysis showed low downstream metabolites vanillylmandelic acid, homovanillic acid but high 3-O-methyl-L-DOPA, 5-hydroxytryptophan. Cerebrospinal fluid pterins were normal. Genotyping in DDC confirms the diagnosis. Urine organic acid analysis provided the first clue to diagnosis in four of the cases, which then triggered cerebrospinal fluid neurotransmitter and genetic analysis. We also developed a diagnostic decision support system to assist the interpretation of the mass spectrometry data from urine organic acids. CONCLUSIONS: Urine organic acid could be essential in guiding subsequent investigations for the diagnosis of aromatic L-amino acid decarboxylase deficiency. We propose to screen suspected cases first with urine organic acids, specifically looking for vanillactic acid and N-acetylvanilalanine. Suggestive findings should be followed with target analysis for c.714 + 4A > T in ethnically Chinese patients. The assistive tool allowed expedite interpretation of profile data generated from urine organic acids analysis. It may also reduce interpreter's bias when peaks of interest are minor peaks in the spectrum.

In-house multiplex ligation-dependent probe amplification assay for citrin deficiency: analytical validation and novel exonic deletions in SLC25A13.

Citrin deficiency is one of the most common inborn errors of metabolism in East Asians, which may manifest as neonatal cholestasis, failure to thrive and dyslipidaemia, or recurrent hyperammonaemic encephalopathy. Its molecular diagnosis requires confirmation of the presence of biallelic pathogenic variants in SLC25A13 gene by sequencing, and analysis for a common insertion IVS16ins3kb. However, patients with compatible biochemical features but only one monoallelic pathogenic variant have remained a diagnostic challenge. Here we report the development, validation and application of a multiplex ligation-dependent probe amplification (MLPA) assay using an in-house oligonucleotide probemix and a customised Coffalyer.NET worksheet for detection of exonic copy number variations in SLC25A13. With this MLPA assay, we successfully identified the presence of a heterozygous exonic deletion in SLC25A13 in three of 15 (20%) unrelated individuals with only one monoallelic pathogenic variant detected using conventional methods. Three exonic deletions, two novel involving exon 14 and one reported involving exon 5, were subsequently confirmed with Sanger sequencing. In summary, we developed, evaluated, and demonstrated the clinical utility of an in-house MLPA assay to look for exonic deletions in SLC25A13 in patients with citrin deficiency. With the discovery of novel deletions, MLPA should be considered a test of choice for molecular diagnosis of citrin deficiency when the sequencing result is inconclusive.

Toxicity from illegitimate slimming agents - a 10-year case series at a tertiary toxicology laboratory in Hong Kong.

CONTEXT: This retrospective case-series study aims to provide an overview of the clinical, biochemical and analytical findings in patients who presented with toxicity related to the use of illegitimate slimming agents in Hong Kong from the perspective of a tertiary referral toxicology laboratory. METHODS: All clinical cases referred to the Hospital Authority Toxicology Reference Laboratory, Hong Kong with clinical suspicion of illegitimate slimming agent-related toxicity between January 2008 and December 2017 were reviewed retrospectively. The use of illegitimate slimming agents included the use of (1) deregistered slimming agents, (2) drug analogues that were not registered drugs, (3) registered drugs not approved for the indication of weight reduction (whether prescribed by a doctor or not), and (4) prescription-only slimming agents without a doctor's prescription. Patients taking registered weight-reducing drugs prescribed by a doctor were excluded. Patient demographics, clinical features, relevant laboratory investigations, and toxicological findings were analyzed. RESULTS: From 2008 to 2017, a total of 346 patients were analytically confirmed by our laboratory to have clinical toxicity related to the use of illegitimate slimming agents. The median age of the patients was 27 years and 92.5% of the patients were female. The most common clinical presentations included psychiatric features, sympathomimetic toxicity, hypokalemia, and abnormal thyroid function tests. Fatal or severe clinical toxicity was observed in 10% of the cases. The major classes of drugs detected on our analytical platforms were stimulants (e.g., sibutramine), laxatives (e.g., anthraquinones), diuretics (e.g., hydrochlorothiazide), and thyroid hormones (e.g., animal thyroid tissue). These illegitimate slimming agents were obtained from various sources including the Internet, over-the-counter in community pharmacy, or unspecified local sources. DISCUSSION AND CONCLUSIONS: The use of slimming agents is common worldwide; apart from taking registered slimming agents prescribed by registered practitioners, many users obtain slimming agents from various illegitimate sources. The unregulated use of these drugs can be associated with significant clinical toxicity. This study provides a current landscape of illegitimate slimming agent toxicity in Hong Kong to frontline clinicians and other toxicology professionals. Collaboration between clinicians, laboratories, and government authorities would be imperative to prevent further health adversities related to the misuse of these agents.

Superwarfarin (Long-Acting Anticoagulant Rodenticides) Poisoning: from Pathophysiology to Laboratory-Guided Clinical Management.

Superwarfarins are long-acting anticoagulant rodenticides developed from warfarin. The mechanism of action is by inhibition of vitamin K epoxide reductase, resulting in the inability of the body to recycle vitamin K. Deficiency of vitamin K thereafter leads to inability for the body to synthesise vitamin K-dependent coagulation factors, factor II, VII, IX, and X, leading to prolonged prothrombin time. Due to the bulky aromatic sidechains, superwarfarins have a much longer half-life when compared to warfarin, and exposure to superwarfarins results in a prolonged period of anticoagulation which can result in clinical bleeding. Diagnosis is straightforward in patients with known history of superwarfarin exposure but has proved difficult for patients who did not report superwarfarin intake. Superwarfarin poisoning should therefore be suspected in all patients with unexplained prolongation of prothrombin time, and can be confirmed by their detection in serum. Treatment for superwarfarin poisoning includes rapid correction of factor deficiencies with either 4-factor prothrombin complex concentrate or fresh frozen plasma in patients with active bleeding, and high dose vitamin K therapy given multiple times per day for a prolonged period of weeks to months.

Clinical Mass Spectrometry in the Bioinformatics Era: A Hitchhiker's Guide.

Mass spectrometry (MS) is a sensitive, specific and versatile analytical technique in the clinical laboratory that has recently undergone rapid development. From initial use in metabolic profiling, it has matured into applications including clinical toxicology assays, target hormone and metabolite quantitation, and more recently, rapid microbial identification and antimicrobial resistance detection by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this mini-review, we first succinctly outline the basics of clinical mass spectrometry. Examples of hard ionization (electron ionization) and soft ionization (electrospray ionization, MALDI) are presented to demonstrate their clinical applications. Next, a conceptual discourse on mass selection and determination is presented: quadrupole mass filter, time-of-flight mass spectrometer and the Orbitrap; and MS/MS (tandem-in-space, tandem-in-time and data acquisition), illustrated with clinical examples. Current applications in (1) bacterial and fungal identification, antimicrobial susceptibility testing and phylogenetic classification, (2) general unknown urine toxicology screening and expanded new-born metabolic screening and (3) clinical metabolic profiling by gas chromatography are outlined. Finally, major limitations of MS-based techniques, including the technical challenges of matrix effect and isobaric interference; and novel challenges in the post-genomic era, such as protein molecular variants, are critically discussed from the perspective of service laboratories. Computer technology and structural biology have played important roles in the maturation of this field. MS-based techniques have the potential to replace current analytical techniques, and existing expertise and instrument will undergo rapid evolution. Significant automation and adaptation to regulatory requirements are underway. Mass spectrometry is unleashing its potentials in clinical laboratories.

Genetic diagnosis of CADASIL in three Hong Kong Chinese patients: A novel mutation within the intracellular domain of NOTCH3.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset hereditary stroke syndrome characterized by recurrent stroke and progressive cognitive impairment caused by NOTCH3 mutations. We report here the clinical and molecular findings of three unrelated Hong Kong Chinese families with CADASIL syndrome. Sanger sequencing of genomic DNA revealed a novel heterozygous variant NM_000435.2(NOTCH3):c.[5903_5904insATAA];[5903_5904=] NP_000426.2:p.(Asp1969∗);(Asp1969=) and two previously reported heterozygous mutations NM_000435.2(NOTCH3):c.[328C>T];[328C=] NP_000426.2:p.[(Arg110Cys)];[(Arg110=)] and NM_000435.2(NOTCH3):c.[580T>A];[580T=] NP_000426.2:p.(Cys194Ser);(Cys194=) in the three families respectively. Molecular basis of CADASIL in these three patients were further established. Genetic analysis provides a reliable method for confirming the diagnosis of CADASIL and enables proper genetic counseling and cascade testing.

Retrospective Study of the Characteristics of Anticoagulant-Type Rodenticide Poisoning in Hong Kong.

INTRODUCTION: Warfarin- and superwarfarin-type anticoagulants are commonly used as rodenticides. Exposure to these agents, especially superwarfarins with long-acting anticoagulant effect, can cause life-threatening coagulopathy in humans. Most superwarfarin poisoning cases had an obvious history of exposure, though occult cases without exposure history have also been reported. The current study aims to examine anticoagulant-type rodenticide poisoning in Hong Kong and to identify the similarities and differences between patients with known exposure history and those whose exposure is recognized only through laboratory testing. METHODS: The present study was conducted in a tertiary referral clinical toxicology laboratory in Hong Kong. This was a retrospective cohort study of all patients with biochemically confirmed anticoagulant-type rodenticide exposure, from 2010 to 2014. RESULTS: Superwarfarin was the most common group of anticoagulant-type rodenticides identified (87.8%), in which bromadiolone and brodifacoum were the most frequently encountered. Among the 41 cases identified, 31 had an obvious exposure history, and 10 were occult poisoning in which the context of exposure remained unidentified. All occult poisoning patients without exposure history presented with bleeding events. These occult poisoning cases often went unrecognized by frontline clinicians, leading to delayed investigation and initiation of treatment. This group of patients was associated with a longer time to diagnose coagulopathy (p < 0.001) and confirm rodenticide poisoning (p < 0.05), a higher rate of international normalized ratio (INR) rebound after initiation of antidote (p < 0.001), and a longer time needed for normalizing INR (p < 0.05). CONCLUSION: Occult superwarfarin poisoning is an important yet under-recognized differential cause of unexplained coagulopathy. A high index of clinical suspicion and availability of specialized toxicological test for superwarfarins play a vital role in diagnosis and early initiation of appropriate management. The underlying cause of such poisoning remains obscure and warrants further study.

Adulteration of proprietary Chinese medicines and health products with undeclared drugs: experience of a tertiary toxicology laboratory in Hong Kong.

AIMS: Proprietary Chinese medicines (pCMs) and health products, generally believed to be natural and safe, are gaining popularity worldwide. However, the safety of pCMs and health products has been severely compromised by the practice of adulteration. The current study aimed to examine the problem of adulteration of pCMs and health products in Hong Kong. METHODS: The present study was conducted in a tertiary referral clinical toxicology laboratory in Hong Kong. All cases involving the use of pCMs or health products, which were subsequently confirmed to contain undeclared adulterants, from 2005 to 2015 were reviewed retrospectively. RESULTS: A total of 404 cases involving the use of 487 adulterated pCMs or health products with a total of 1234 adulterants were identified. The adulterants consisted of approved drugs, banned drugs, drug analogues and animal thyroid tissue. The six most common categories of adulterants detected were nonsteroidal anti-inflammatory drugs (17.7%), anorectics (15.3%), corticosteroids (13.8%), diuretics and laxatives (11.4%), oral antidiabetic agents (10.0%) and erectile dysfunction drugs (6.0%). Sibutramine was the most common adulterant (n = 155). The reported sources of these illicit products included over-the-counter drug stores, the internet and Chinese medicine practitioners. A significant proportion of patients (65.1%) had adverse effects attributable to these illicit products, including 14 severe and two fatal cases. Psychosis, iatrogenic Cushing syndrome and hypoglycaemia were the three most frequently encountered adverse effects. CONCLUSIONS: Adulteration of pCMs and health products with undeclared drugs poses severe health hazards. Public education and effective regulatory measures are essential to address the problem.

Supraventricular tachycardia and acute confusion following ingestion of e-cigarette fluid containing AB-FUBINACA and ADB-FUBINACA: a case report with quantitative analysis of serum drug concentrations.

BACKGROUND: AB-FUBINACA and ADB-FUBINACA are structurally similar synthetic cannabinoids with potent CB1 receptor agonistic effects. Very little is known about their pharmacology and toxicology. OBJECTIVE: To report a case of supraventricular tachycardia and acute confusion after ingestion of e-cigarette fluid containing AB-FUBINACA and ADB-FUBINACA, with quantitative analysis of the serum drug concentrations. CASE REPORT: A healthy 24-year-old man ingested two drops of e-cigarette fluid which were later found to contain AB-FUBINACA and ADB-FUBINACA. Within 30 min of ingestion, he became somnolent, confused, and agitated, with palpitation and vomiting. On arrival to the emergency department, a short run of supraventricular tachycardia was noted, which resolved spontaneously. Bedside urine immunoassay failed to detect recreational drugs. Laboratory blood tests showed mild hypokalemia. Exposure to AB-FUBINACA and ADB-FUBINACA was confirmed analytically, with serum concentrations of 5.6 ng/mL and 15.6 ng/mL, respectively, in the blood sample collected on presentation. The patient recovered uneventfully with supportive treatment and was discharged 22 h after admission. DISCUSSION: AB-FUBINACA and ADB-FUBINACA are orally bioavailable with rapid onset of toxicity after ingestion. In this case, supraventricular tachycardia was likely the result of exposure to AB-FUBINACA and ADB-FUBINACA. The serum concentrations of AB-FUBINACA and ADB-FUBINACA were higher than those previously reported in fatal cases. CONCLUSION: In the context of acute poisoning, the presence of unexplained tachyarrhythmias, confusion, and a negative recreational drug screen should prompt clinicians to consider synthetic cannabinoid toxicity as a differential diagnosis.

Bi-variate approach to negative interference of bilirubin towards an acetaminophen assay.

BACKGROUND: It is well known that enzymatic assays for acetaminophen are positively interfered by bilirubin. The effect on acetaminophen not only depends on the concentration of bilirubin but also on that of acetaminophen. We demonstrated a negative interference instead of a positive one in a commonly used routine analyzer and investigated the recovery of acetaminophen in an enzymatic assay by a bi-variate regression. METHODS: Commercially available blank serum specimens were spiked with acetaminophen and bilirubin at various concentrations, and were analyzed in the Beckman Coulter AU5822 analyzer. The specimens were run in duplicates. The results were then analyzed by least-square analysis and was built into a bi-variate quadratic model. RESULTS: The recovery of acetaminophen in this experiment ranged from 38.9% to 100% throughout a range of 23 µmol/L to 2052 µmol/L (for acetaminophen) and 19 µmol/L to 570 µmol/L (for bilirubin). A contour map, as well as a bi-variate equation was established, describing the relationship between acetaminophen recovery, acetaminophen concentration, and bilirubin concentration. CONCLUSION: It was shown that the degree of bilirubin interference in a commercially available acetaminophen assay is dependent on both bilirubin and acetaminophen concentrations. There was a decrease in the apparent acetaminophen concentration by an average of 30% at a bilirubin concentration of 420 µmol/L in the Beckman Coulter AU5822 analyzer. The complex relationship can be modeled by mathematical means. This allows the laboratory staff to estimate the recovery of acetaminophen when bilirubin level is concurrently measured.

Dystroglycanopathy with two novel POMT1 mutations in a Chinese boy with developmental delay and muscular dystrophy.

Alpha-dystroglycanopathies are a group of diseases due to reduced glycosylation of alpha-dystroglycan, which commonly result from mutations in POMT1, POMT2, and POMGnT1. Patients with alpha-dystroglycanopathies present with muscular, cerebral, and ocular involvements with differing severities. We reported a boy who presented with muscular dystrophy, developmental delay, and non-specific white matter lesions. Mutation analysis of POMT1 was performed and revealed two novel mutations, a substitution mutation (c.176T>G) and a duplication mutation (c.2059dupC) which results in premature termination of translation. In-silico prediction in five different platforms concurred that the substitution is damaging, and functional studies by immunofluorescence revealed lack of staining in the carbohydrate moiety of alpha-dystroglycan, confirming the molecular findings in a functional manner. In conclusion, we reported the first case of genetically confirmed alpha-dystroglycanopathy due to mutations in POMT1 in Chinese.

Alpha-2 agonists in acute pain management.

IMPORTANCE OF THE FIELD: This review explores the significance of alpha-2 agonists used clinically in acute pain management. AREAS COVERED IN THIS REVIEW: Although alpha-2 agonists have been reported to have an analgesic effect, they are not commonly used clinically for acute pain management. Clinical studies on use of alpha-2 agonists for acute pain management are reviewed and discussed. A literature search was done using Medline with the keywords 'alpha-2 agonist', 'clonidine', 'dexmedetomidine', 'fadolmidine', 'pharmacokinetics', 'pharmacodynamics', 'postoperative analgesia', 'epidural', 'intrathecal', 'peripheral nerve block' and various combinations with these keywords. The years 1977 - 2009 have been included, with particular focus on clinical studies from between 1990 and 2009. WHAT THE READER WILL GAIN: This article helps to clarify the clinical use of alpha-2 agonists in acute pain management according to current, up-to-date evidence. Clinically, available alpha-2 agonists, including clonidine and dexmedetomidine, are discussed in detail. TAKE HOME MESSAGE: Alpha-2 agonists, especially clonidine, seem to be promising with regard to acute postoperative pain management. However, more clinical evidence on dexmedetomidine is necessary to confirm its definite role in acute postoperative pain control.