Use of liquid chromatography-tandem mass spectrometry for determination of higenamine in urine following oral administration of traditional Chinese medicine

Since higenamine (HG) was first included in the World Anti-doping Agency (WADA) 2017 Prohibited List, an increasing number of plants have been found to contain this ingredient. As a result, doctors are hesitant to prescribe traditional Chinese medicine (TCM) to athletes. Thus, it is very important to assess the risks of doping violations due to HG following the oral administration of TCM. We determined the drug concentration-time curves for HG in urine by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after single or multiple administrations of lotus seed powder on volunteers, the single dose was equivalent to 750 μg of HG, and the multiple doses were equivalent to 90 μg of HG each, 3 times daily for 5 consecutive days. For the single-dose group, the HG could be detected in urine 0.5 h after administration and reached a maximum concentration of 16.5 ng/mL 1 h after administration. For the multiple-dose group, the HG concentrations in urine showed two peaks at 29 and 77 h post-administration with 22.6 and 23.1 ng/mL, respectively. At the dosage used in this study, the maximum concentration of HG in some urine samples exceeded the WADA limit of 10.0 ng/mL; the risk was still very high, so athletes must avoid this amount of HG when using TCM. In addition, our study provided further data supporting the presence of sulfonated metabolites of HG in urine samples.     

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction–tandem mass spectrometry to detect hCG use

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti‐Doping Agency (WADA) list of prohibited substances. The majority of WADA‐accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study we measured the concentration of intact hCG, free β‐subunit (hCGβ) and β‐subunit core fragment (hCGβcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC–MS/MS method. Mean concentrations of intact hCG, hCGβ and hCGβcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5^th percentile) for intact hCG, hCGβ and hCGβcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.     

The risk of higenamine adverse analytical findings following oral administration of plumula nelumbinis capsules

Higenamine is a β2‐agonist that has been included in the Prohibited List of the World Anti‐Doping Agency (WADA) since 2017. Meanwhile, it exists in plumula nelumbinis, a part of the lotus seed, and is commonly used as an ingredient in cuisines, herbal medicines, and nutritional supplements in China and other countries in East Asia. Therefore, an evaluation of the risk of an adverse analytical finding (AAF) of higenamine caused by plumula nelumbinis products is necessary in doping control. In this study, 14 volunteers took plumula nelumbinis capsules orally (0.34 g/caplet, 6 caplets/day, 7 days), and another 11 volunteers ingested higenamine tablets (three 5 mg tablets/day for 7 days). Urine samples were collected over a period of 14 days. All urine samples were subjected to quantitative dilute‐and‐shoot analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytical results showed that urinary higenamine concentrations exceeded the WADA reporting limit (10 ng/mL) during the drug period in most sample groups. The maximum higenamine concentration observed in the plumula nelumbinis capsule group was 500 ng/mL. Based on confidence interval theory, appropriate data were used to establish mathematical models. The models reflected that the higenamine concentration in urine can exceed the WADA reporting limit with a high probability after taking plumula nelumbinis capsules. In conclusion, oral administration of plumula nelumbinis capsules showed a high risk of an AAF due to higenamine.     

Elimination profiles of betamethasone after different administration routes: Evaluation of the reporting level and washout periods to ensure safe therapeutic administrations

Betamethasone (BET) is prohibited in sports competitions when administered by systemic routes, and it is allowed by other routes for therapeutic purposes. In out-of-competition periods, there is no restriction of use. The present work aimed to assess the urinary excretion of BET and its metabolites after allowed and prohibited administrations to verify the suitability of the current reporting level of 30 ng/ml used to distinguish allowed and prohibited administrations and to establish washout periods for oral and intramuscular (IM) administrations when out-of-competition treatments are needed. BET was administered to healthy volunteers by different routes: topical (10 mg/day for 5 days, n = 6 males), intranasal (320 μg/day for 3 days, n = 4 males and 4 females), oral (0.5 mg, n = 8 males) or IM (6 mg, n = 6 males, or 12 mg, n = 4 males and 4 females). Urine and plasma samples collected before and after administration were analysed using liquid chromatography–tandem mass spectrometry. Among all studied metabolites, the parent drug was selected as the best discriminatory marker. After topical administration, BET concentrations were lower than 6.6 ng/ml. However, after intranasal treatment, some samples at concentrations close to or higher than 30 ng/ml were detected, suggesting the need to revise the current reporting level. Urinary concentrations after oral and intranasal administrations were similar, and after IM administration, concentrations were much higher. Taking into account all information, a urinary reporting level of 60 ng/ml is proposed. Washout periods of at least 48 and 96 h are recommended after oral and IM administrations, respectively.     

Acute and subacute oral toxicity of periodate salts in rats

Periodate salts are being developed as potential replacements for perchlorate due to potential health hazards associated with exposure to perchlorate. The aim of this study was to investigate acute and subacute effects of periodate salts in rats. Acute oral toxicity of potassium and sodium periodate was determined using the Sequential Stage-Wise Probit method. The LD₅₀ for potassium periodate was 732 (95% CI = 539–838, slope = 13.4) and 685 mg/kg (95% CI = 580–809, slope = 10.6) for females and males, respectively. The LD₅₀ for sodium periodate was 318 (95% CI = 292–347, slope = 24.3) and 741 mg/kg (95% CI = 704–779, slope = 31.2) for females and males, respectively. In the subacute study, rats were administered sodium periodate at five doses (1/16 LD₅₀ up to LD₅₀) or distilled water for 14-days via oral gavage. Female rats in the 318 mg/kg-day group and male rats in the 185, 370, and 741 mg/kg-day groups exhibited moribundity, kidney toxicity, uremia, and a stress response. BMDL₁₀s of 17.2 and 33.7 mg/kg-day were derived for females and males, respectively. Comparison with the NOAEL for perchlorate-induced thyroid toxicity in rats (0.009 mg/kg-day) suggests sodium periodate is less toxic than perchlorate on a subacute basis.     

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements

Diuretic agents are prohibited in sports in‐ and out‐of‐competition according to the regulations of the World Anti‐Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA‐accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ‐contaminated powder (6.4 μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid–liquid extraction and liquid chromatography–tandem mass spectrometry determination. Post‐administration specimens were found to contain HCTZ at concentrations of 5–230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.     

Detection of cocaine and its metabolites in whole blood and plasma following a single dose,controlled administration of intranasal cocaine

The disposition of drugs and their metabolites have been extensively described in the literature, based primarily on the analysis of plasma and urine. However, there are more limited data on their disposition in whole blood, which is often the only specimen available in forensic investigations and cases of driving under the influence of drugs. In this study, we have, for the first time, established pharmacokinetic properties of cocaine (COC) and its metabolites from concurrently collected whole blood and plasma samples, following a single 100 mg dose of cocaine hydrochloride administered via nasal insufflation to seven healthy volunteers. The median C_max of COC and its major metabolites, benzoylecgonine (BZE) and ecgonine methyl ester (EME), were closely related in whole blood and plasma. The median C_max for COC in plasma was 379.7 ng/mL (347.5–517.7) and 344.24 ng/mL (271.6–583.2) in whole blood. The median C_max for BZE in plasma was 441.2 ng/mL (393.6–475. and 371.18 ng/mL (371.1–477.3) in whole blood, EME was 105.5 ng/mL (93.6–151.8) in plasma and 135.5 ng/mL (87.8–183) in whole blood. Calculated medians of the whole blood to plasma ratio of COC (0.76), BZE (0.98) and EME (1.02) of approximately 1, strongly suggesting that the erythrocyte cell wall presents no barrier to COC and its metabolites. Furthermore, whole blood and plasma concentrations of COC were strongly correlated (R² = 0.0914 R = 0.956, p < 0.0001), as was BZE (R² = 0.0932 R = 0.965, p < 0.0001) and EME (R² = 0.0964R = 0.928, p < 0.0001). The minor oxidative metabolite norcocaine (NCOC) was detected in both whole blood and plasma at concentrations between 1 and 5 ng/mL within 60–180 minutes, suggesting that NCOC could be indicator of recent COC administration. Data from this study have shown for the first time that COC and its metabolites BZE and EME are evenly distributed between plasma and whole blood following controlled single‐dose intranasal COC administration.     

Urinary bisphenol A and semen quality,the LIFE Study

Bisphenol A (BPA), a high-production volume industrial chemical found in several consumer products, has been negatively associated with sperm quality. This study aimed to estimate the association between BPA and 35 measures of semen quality among reproductive aged men recruited from 16 counties in Michigan and Texas, 2005–2009. Of 501 enrolled males, 418 (83.4%) provided a urine sample and at least one semen sample. Linear and logistic regression models assessed the association between urinary BPA levels and individual semen quality endpoints. Generalized estimating equations were used to account for repeated measures of semen quality and adjusted models accounted for 11 a priori covariates. Geometric mean total urinary BPA concentration among participants was 0.55 ng/mL (95% CI 0.49–0.63). A negative relation between BPA and DNA fragmentation was the sole significant finding in adjusted linear regression (β = −0.0544, p = 0.035) and suggestive of less sperm DNA damage.     

Evaluation of apomorphine HCl effects on reproductive endpoints: studies in male rats and dogs.

Apomorphine HCl is currently in phase III clinical trials for the treatment of erectile dysfunction. The potential for reproductive toxicity in males was assessed based on a 13-week rat study--a fertility study in male rats--and a 6-month study in dogs. The subcutaneous (s.c.) route was selected to simulate the sublingual route in humans. Dosages of apomorphine were 0.0 (vehicle), 0.8, 2, and 8 mg/kg/day in the 13-week study in rats (20/group), with 8 mg/kg/day used for only 9 weeks. In the fertility study, 24 males/group were cohabited with females, and doses were 0.0, 0.2, 0.8, and 2 mg/kg/day. Males were treated for 4 weeks prior to cohabitation and for 5 weeks throughout cohabitation. Organ weights, including testis and left epididymis, sperm count and morphology in the epididymis, and sperm motility in the vas deferens were evaluated. Male fertility index, and in females, the numbers of fetuses, implantation sites, and corpora lutea were counted. Male dogs (five/group) were dosed with 0.04, 0.1, or 0.4 mg/kg/day for 6 months. Epididymal and prostate weight, and testicular and epididymal histology were evaluated. Daily morbidity/mortality, weekly clinical observations, body weight, and food consumption were evaluated in all studies. No adverse effect was observed in any of the reproductive parameters in the studies. The NOEL for reproductive toxicity was approximately equal to 0.4 mg/kg/day in dogs and > or = 2 mg/kg/day in rats. These doses in rats and dogs correlated with plasma levels of approximately 240 and 130 ng/mL, and AUCs of 200 and 100 ng.h/mL, respectively. These levels suggest a safety margin for the evaluated male reproductive endpoints of at least 104 times based on the Cmax, and 44 times based on AUC of the clinical dose.     

Assessment of the efficacy of drug transdermal delivery by electro-phonophoresis in treating tuberculous lymphadenitis

Abstract

Electro-phonophoresis (EP) has been used in various clinical fields. The objective of present study is to evaluate the skin permeability of isoniazid (INH) and rifampicin (RIF) in patients with tuberculous lymphadenitis with the aid of EP to validate the clinical applications of this transdermal delivery system for the treatment of superficial extrapulmonary tuberculosis. INH and RIF solutions were delivered transdermally, with or without EP, in the surrounding tissue of the lesion for 0.5 h. Local pyogenic fluids or necrotic tissue samples from the infection sites in patients were collected at 1 h after dosing. Drug concentrations in samples were evaluated by high performance liquid chromatography. The median INH and RIF intra-lesional concentrations were 0.365 (interquartile range [IQR] 0.185–1.775) μg/mL and 1.231 (IQR 0.304–1.836) μg/mL in oral group; 2.964 (IQR 0.193–7.325) μg/mL and 2.646 (IQR 1.211–3.753) μg/mL in INH- and RIF-transdermal plus EP group. Drug concentrations in the local sites of patients receiving INH or RIF through EP transdermal delivery were statistically higher than those observed in patients only taking INH and RIF orally. However, this enhancement was not observed in the transdermal delivery of INH or RIF without EP in contrast to the oral administrations of drugs. EP can effectively enhance the skin permeability of INH and RIF in patients with tuberculous lymphadenitis. The increase in drug concentrations in the lesions could help eradication of the germs; shorten the treatment course and increase the cure rate of patients with tuberculous lymphadenitis.     

Determinants of community pharmacists’ information gathering and counseling practices during the management of minor ailments

ObjectiveTo assess the determinants of community pharmacists’ information gathering and counseling practices during the management of minor ailments in Qatar.MethodA cross-sectional study of 305 community pharmacists was conducted with a pre-tested 27-item questionnaire. Bivariate logistic regression was used to identify the determinants of information gathering and counseling practices.ResultsThe response rate was 92.5% (282/305). A majority of the respondents (68.1%) were males, within the age range of 31–40 years (55.3%), work for chains pharmacies (77.3%), and were predominantly of foreign nationalities (94.7%). Patients’ identity (91.1%), age (92.2%), symptoms (92.6%) and duration of symptoms (89.3%) were most frequent information gathered, while dose (99%), frequency (97.8%), route of administration (95.7%), and duration of use (92.9%) were the most frequent counseling information. Median information gathering score was significantly higher in females and among community pharmacists in chain pharmacies (p < 0.05), while median counseling practice scores were significantly higher among in chain pharmacies (p < 0.05). Consultation time of 6–10 min (OR = 1.75, 95% CI: 1.02–3.0, p = 0.04) and female gender (OR = 2.10, 95% CI: 1.16–3.79, p = 0.01) were significant determinants of information gathering, while age group (31–40 years) (OR = 1.84, 95% CI: 1.05–3.22, p = 0.03) and consultation time (6–10 min) (OR = 2.24, 95% CI: 1.31–3.86, p = 0.003) were significant determinants of counseling practices.ConclusionThe significant determinants of community pharmacists’ Information gathering and counseling practices during the management of minor ailments were female gender and consultation time (6–10 min), and age group (31–40 years) and consultation time (6–10 min) respectively.     

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti‐Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO_2max and a 20‐km time‐trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0–24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol C_max was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·?h) than EX (16.9±2.9 ng/mL·?h)(P<0.01) and EXD (16.1±3.2 ng/mL·?h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative prediction of hepatic CYP3A activity using endogenous markers in healthy subjects after administration of CYP3A inhibitors or inducers

Accurate prediction of cytochrome P450 (CYP) 3A activity in the early stage of drug development and in clinical practice is important. This study aimed to evaluate the previously constructed CYP3A activity prediction model after administration of CYP3A inhibitors and inducers and to modify the model for better prediction of CYP3A activity. Healthy male subjects received the following study drugs during three study periods: midazolam alone (control phase); midazolam with 200 mg of itraconazole (CYP3A inhibition phase); and midazolam with 150 mg of rifampicin (CYP3A induction phase). We quantified the concentrations of several endogenous CYP3A markers in both urine and plasma using gas chromatography-mass spectrometry. The urinary markers, including 6β-hydroxy (OH)-cortisol/cortisol, 6β-OH-cortisone/cortisone, 16α-OH-dehydroepiandrosterone (DHEA)/DHEA, 16α-OH-androstenedione (A-dione)/A-dione and 7β-OH-DHEA/DHEA, were significantly correlated with midazolam clearance in both the CYP3A inhibition and induction phases. We constructed a statistical prediction model after integrating data from a previous study to predict midazolam clearance as follows: Ln(midazolam clearance) = 2.5545 + 0.3988 × ln(7β-OH-DHEA/DHEA) + 0.1984 × ln(16α-OH-DHEA/DHEA) + 0.5031 × ln(6β-OH-cortisol/cortisol) – 0.1261 [ln(7β-OH-DHEA/DHEA) × ln(6β-OH-cortisol/cortisol)] (r² = 0.75). We suggest that quantitating endogenous markers in vivo coupled with the statistical prediction model may be useful for predicting CYP3A parameters.     

PEth 16:0/18:1 and 16:0/18:2 after consumption of low doses of alcohol—A contribution to cutoff discussion

Phosphatidylethanol in blood has gained recognition as a direct alcohol biomarker. Although different cutoffs have been suggested, there is no consensus for differentiating abstinence from alcohol consumption. In this study, 75 participants (72% female) consumed 20 g of ethanol on three consecutive evenings. Blood was sampled on each following day and PEth 16:0/18:1 and 16:0/18:2 were determined. PEth 16:0/18:1 ranged from 8.9–21.5, 8.7–19.3, and 8.8–42.3 ng/ml and PEth 16:0/18:2 from 8.7–31.7, 9.0–39.3, and 9.4–43.0 ng/ml after the respective days of ethanol consumption. PEth 16:0/18:1 yielded a sensitivity of 25%, 45%, and 49% and PEth 16:0/18:2 of 40%, 61%, and 68% for the consumption days, respectively (cutoff 10 ng/ml). PEth 16:0/18:1 reached >20 ng/ml in five samples overall. Sensitivity of PEth 16:0/18:2 > 20 ng/ml was better with 35% after the three drinking days. Overall, PEth 16:0/18:1 was >35 ng/ml in one sample and PEth 16:0/18:2 in three samples. Significantly, more women had PEth 16:0/18:1 > 10 ng/ml after the third day of consuming 20 g of alcohol (p = 0.02) and PEth 16:0/18:2 > 10 ng/ml after the second (p = 0.023) and the third (p = 0.002) consumption, which can be led back to the higher blood alcohol concentration women reach after consuming the same alcohol amount as men. Although the response rates of PEth to alcohol uptake are subject to strong interindividual differences, results suggest that PEth cutoff should be lowered for better detection of consumption of low to medium amounts of alcohol. Furthermore, it is advantageous to analyze both PEth 16:0/18:2 and 16:0/18:1.     

Range of therapeutic prothipendyl and prothipendyl sulfoxide concentrations in clinical blood samples

Due to a lack of reference blood concentrations in the literature, the forensic evaluation of prothipendyl findings in blood samples is difficult. Interpretations with regard to the assessment of blood concentrations as well as an estimation of the ingested prothipendyl amounts were often vague. To describe a concentration range in clinical samples, prothipendyl and prothipendyl sulfoxide concentrations were determined in serum samples of 50 psychiatric patients receiving 40 mg, 80 mg, or 160 mg doses of prothipendyl. The analyses of prothipendyl and prothipendyl sulfoxide were carried out using validated methods of high performance liquid chromatography coupled to triple quadrupole mass spectrometry (LC–QQQ–MS), respectively. 40 mg doses caused average prothipendyl serum concentrations of 18.0 ng/mL (1 hour after intake) and 7.9 ng/mL (10.5 hours after intake), while 80 mg doses caused averages of 42.6 ng/mL and 15.2 ng/mL at the mentioned times of sampling. Irrespective of the given dose, prothipendyl concentrations below 30 ng/mL were observed in 80% of the patient samples taken 1 hour after ingestion as well as in 90% of the samples collected 10.5 hours after administration. Serum concentrations of the Phase I metabolite prothipendyl sulfoxide averaged 4.3 ng/mL (1 hour after intake) and 3.6 ng/mL (10.5 hours after intake). Possible drug‐drug interactions regarding absorption and metabolism of prothipendyl are discussed. Results of the herein presented study are useful for the interpretation of analytical prothipendyl findings in forensic toxicology. The utility of the described concentration range is demonstrated by discussing two death cases involving prothipendyl findings.     

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS

IntroductionHepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia.MethodsAn LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays.ResultsThe calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at ? 60 to ? 80 °C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were ? 11.2% and 5.6%, respectively at the LLOQ, and less than ± 7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between ? 0.7 and + 2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n = 16, median 121 ng/mL) and 99 ng/mL (n = 50, median 68 ng/mL), respectively.ConclusionsA fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples.     

Additional role of serum 25‐hydroxyvitamin D3 levels in atherosclerosis in Chinese middle‐aged and elderly men

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Response surface modelling of the pharmacodynamic interaction between propofol and remifentanil in patients undergoing anaesthesia

This study describes the pharmacodynamic interaction between propofol and remifentanil. Sixty patients who were scheduled for elective surgery under general anaesthesia (30 males/30 females) were enrolled. Patients were randomly allocated to receive one of 15 combinations of drug levels. Baseline electroencephalograms (EEGs) were recorded for 5 minutes prior to administering the drugs. Patients received a target‐controlled infusion at one of four predefined doses of propofol (high, 3 μg/mL; medium, 1.5 μg/mL; low, 0.5 μg/mL; or no drug) and of remifentanil (high, 6 or 8 ng/mL; medium, 4 ng/mL; low, 2 ng/mL; or no drug). The occurrence of muscle rigidity, apnoea, and loss of consciousness (LOC) was monitored, and EEGs were recorded during the drug administration phase. Electroencephalographic approximate entropy (ApEn) and temporal linear mode complexity (TLMC) parameters at baseline and under steady state conditions were calculated off‐line. Response surfaces were developed to map the interaction between propofol and remifentanil to the probability of occurrence for quantal responses (muscle rigidity, apnoea, LOC) and ApEn and TLMC measurements. Model parameters were estimated using non‐linear mixed effects modelling. The response surface revealed infra‐additive and synergistic effects for muscle rigidity and apnoea, respectively. The effects of the combined drugs on LOC and EEG parameters (eg, ApEn and TLMC) were additive. The C₅₀ estimates of remifentanil (ng/mL) and propofol (μg/mL) were 9.11 and 130 000 for muscle rigidity, 8.99 and 6.26 for apnoea, 13.9 and 3.04 for LOC, 23.4 and 10.4 for ApEn, and 14.8 and 6.51 for TLMC, respectively. The probability of occurrence for muscle rigidity declined when propofol was combined with remifentanil.     

Enantioselective disposition of (R,R)‐formoterol, (S,S)‐formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control

Formoterol is a long‐acting beta2‐adrenoceptor agonist (LABA) used for the treatment of asthma and exercise‐induced bronchoconstriction. Formoterol is usually administered as a racemic (rac‐) mixture of (R,R)‐ and (S,S)‐enantiomers. While formoterol is restricted by the World Anti‐Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)‐formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac‐formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) assay. Total (free drug plus conjugated metabolite) median (min‐max) rac‐formoterol urine levels following inhalation were 1.96 (1.05–13.4) ng/mL, 1.67 (0.16–9.67) ng/mL, 0.45 (0.16–1.51) ng/mL, 0.61 (0.33–0.78) ng/mL, and 0.17 (0.08–1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)‐formoterol (around 30%–60% of total) compared to (S,S)‐formoterol (0%–30%). There was clear evidence of inter‐individual enantioselectivity observed in the ratios of (R,R):(S,S)‐formoterol, where (S,S)‐ was predominant in free formoterol, and (R,R)‐ predominant in the conjugated metabolite. In conclusion, rac‐formoterol delivered by inhalation exhibits enantioselective elimination in urine following single‐dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2‐agonist chiral switch products such as (R,R)‐formoterol, and total hydrolyzed rac‐formoterol is warranted to account for inter‐individual differences in enantioselective glucuronidation.