Doping control analysis of myo-inositol trispyrophosphate and 10 bisphosphonates in equine plasma by ion chromatography-mass spectrometry and its application to clodronic acid horse administration.

Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as since February 2019, the International Agreement on Breeding, Racing and Wagering (IABRW) by the International Federation of Horseracing Authorities (IFHA) had identified specific conditions on which bisphosphonates should not be administered to a racehorse. A recent review of literature shows that there is yet a simultaneous screening method for detecting ITPP and bisphosphonates in equine samples. This paper describes an efficient ion chromatography high-resolution mass spectrometry (IC-HRMS) method for the simultaneous detection of ITPP and 10 bisphosphonates at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE) and its application to an administration study of clodronic acid in horses.

Identification of erythropoietin mimetic peptide 1 linear form in a sealed vial and its administration study in horses for doping control purpose.

The erythropoietin mimetic peptide 1 linear form (EMP1-linear), GGTYSCHFGPLTWVCKPQGG-NH2 , was identified in an unknown preparation consisting of white crystalline powder contained in sealed glass vials using ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). The white crystalline powder, allegedly used for doping racehorses, was found to contain around 2% (w/w) of EMP1-linear. EMP1-linear can be cyclised in equine plasma at physiological temperature of 37°C by forming an intramolecular disulfide bond to give EMP1, which is a well-known erythropoiesis stimulating agent that can bind to and activate the receptor for cytokine erythropoietin (EPO). Thus, EMP1-linear is a prodrug of EMP1, which is a performance-enhancing doping agent that can be misused in equine sports. In order to identify potential target(s) for detecting the misuse of EMP1-linear in horses, an in vitro metabolic study using horse liver S9 fraction was performed. After incubation, EMP1-linear mainly existed in its cyclic form as EMP1, and four N-terminus truncated in vitro metabolites TYSCHFGPLTWVCKPQGG-NH2 (M1), SCHFGPLTWVCKPQGG-NH2 (M2), WVCKPQGG-NH2 (M3) and VCKPQGG-NH2 (M4) were identified. An intravenous administration study with the preparation of white crystalline powder containing EMP1-linear was also conducted using three retired thoroughbred geldings. EMP1 was detectable only in the postadministration plasma samples, whereas the four identified in vitro metabolites were detected in both postadministration plasma and urine samples. For controlling the misuse of EMP1-linear in horse, its metabolite M3 gave the longest detection time in both plasma and urine and could be detected for up to 4 and 27 h postadministration, respectively.

Patient-reported health-related quality of life outcomes following mastectomy for breast cancer, with immediate, delayed or no breast reconstruction: Four-year follow-up from a prospective cohort study.

BACKGROUND: Breast reconstruction (BR) improves women's health-related quality of life (HRQOL) following mastectomy for breast cancer, yet factors contributing to improved HRQOL remain unclear. This study aimed to explore the overall impact of mastectomy with or without BR on participants' perceptions of HRQOL over time in a cohort of women with high-risk breast cancer; to examine differences in mean HRQOL scores between immediate BR, delayed BR and no BR groups; to assess the influence of patient characteristics potentially associated with HRQOL scores; and to determine the feasibility of long-term collection of patient-reported outcome measures in clinical settings. METHODS: A prospective, longitudinal study of 100 women with high-risk breast cancer who underwent mastectomy with or without breast reconstruction and were likely to require post-mastectomy radiotherapy. Four validated patient-reported questionnaires, comprising 21 outcome measures relating to HRQOL, administered at baseline and up to 4 years post-mastectomy. Demographic, clinical and surgical data extracted from patient medical records. RESULTS: Consistently significant declines in perceptions of future health and arm symptoms, consistently significant improvements in treatment side effects, breast symptoms and fatigue, as well as significant improvements, compared to baseline, in social functioning and financial difficulties at 48 months. No significant differences in mean HRQOL scores between women given a choice of reconstructive options. CONCLUSION: Similar trajectories of HRQOL scores were found in women with high-risk breast cancer who were offered a choice of BR. Informed choice may be an independent contributing factor in long-term maintenance of most HRQOL indicators at their pre-mastectomy levels.

The role of breast reconstruction choice on body image patient-reported outcomes at four years post-mastectomy for breast cancer: A longitudinal prospective cohort study.

OBJECTIVES: To examine the impact of breast reconstruction on women's perceptions of body image over time and to assess the influence of sociodemographic variables on body image. METHODS: A prospective, longitudinal cohort study, using validated breast cancer-specific questionnaires, to compare patient-reported outcomes in women choosing immediate (n = 61), delayed (n = 16) or no (n = 23) breast reconstruction. RESULTS: One hundred women completed baseline questionnaires that included items on body image; 30 women completed all four annual follow-up sets, while 20 women completed baseline only. The three groups were well matched at baseline and similar trajectories in body image measures were identified over 48 months in all groups. At 12 months post-mastectomy, significant changes were seen in eight of the 10 subscales; this reduced to seven subscales at 24 months and four at 36 months. By 48 months, only three subscales remained significantly different to baseline scores: women remained less vulnerable and had fewer limitations (improved outcomes); the one worse outcome was persistently higher levels of arm concern. Three of the sociodemographic variables (health insurance, age and employment status) showed significant inter-group differences at some time points. CONCLUSION: These findings suggest women recover from the negative impact of mastectomy on body image within four years of surgery, whether they have immediate, delayed or no reconstruction. Our results provide some indirect evidence that having a choice of BR options is important, regardless of the choice made. Four years appears to be a suitable follow-up period for future studies in this area.

THE PREVALENCE OF PROBABLE FAMILIAL CHYLOMICRONEMIA SYNDROME IN A SOUTHERN CALIFORNIA POPULATION.

OBJECTIVE: To estimate the prevalence of probable familial chylomicronemia syndrome (FCS) in a major Southern California Academic Center as well as to provide a systematic review of past FCS studies and management recommendations. METHODS: Electronic medical records were queried based on single fasting plasma triglyceride (TG) levels of ≥880 mg/dL and at least 1 episode of acute pancreatitis. After the exclusion of secondary causes (diabetes, alcohol misuse, gallbladder disease, chronic kidney disease, uncontrolled hypothyroidism, estrogen, and drug use) and responses to lipid-lowering treatment, probable patients with FCS were identified. A systematic review of all published literature on the prevalence and management guidelines for FCS was then presented and discussed. RESULTS: Out of 7 699 288 charts queried, 138 patients with TG levels of ≥880 mg/dL and documented evidence of at least 1 episode of acute pancreatitis were identified. Nine patients did not have any documented secondary causes of chylomicronemia. Four of the 9 patients had >20% decrease in TG levels after lipid-lowering treatment, 2 patients were not responsive to lipid-lowering medication, and data on lipid-lowering medications were missing in 3 patients. CONCLUSION: Our study estimates the prevalence of probable FCS at a range of 0.26 to 0.66 per million. Using the recommended criteria, probable FCS cases can be identified to allow early diagnosis and management.

Application of a non-target variable data independent workflow (vDIA) for the screening of prohibited substances in doping control testing.

A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits of the workflow. Additional targets fulfilling the above requirements can be added without changing any settings. The performance of the vDIA workflow was illustrated by applying it to two screening methods in horse urine, with one workflow covering 331 basic drugs and the other covering 45 quaternary ammonium drugs (QADs). Both screening methods have good detection sensitivity with 84% of the basic drugs having Limits of Detection (LoDs) of ≤ 1 ng/mL and 84% of the QADs having LoDs of ≤ 0.4 ng/mL. Other method characteristics including retention reproducibility, method precision and false hit rate will also be presented.

Doping control analysis of antipsychotics and other prohibited substances in equine plasma by liquid chromatography/tandem mass spectrometry.

Antipsychotics are banned substances and considered by the Fédération Equestrian Internationale (FEI) to have no legitimate use in equine medicine and/or have a high potential for abuse. These substances are also prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). Over the years, antipsychotics have been abused or misused in equestrian sports and horseracing. A recent review of literature shows that there is yet a comprehensive screening method for antipsychotics in equine samples. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection of over 80 antipsychotics and other prohibited substances at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE).

Administration study of recombinant human relaxin-2 in horse for doping control purpose.

The insulin-like peptide relaxin (RLX), an endogenous peptide hormone produced in human for pregnancy and reproduction, is also known to exert a range of physiological and pathological effects. Its use is banned in human sports, horseracing, and equestrian competitions due to its potential performance enhancing effect through vasodilation resulting in the increase of blood and oxygen supplies to muscles. Little is known about the biotransformation and elimination of RLX in horses. This paper describes an administration study of rhRLX-2 and its elimination in horses, and the development of sensitive methods for the detection and confirmation of rhRLX-2 in both horse plasma and urine by nano-liquid chromatography/high resolution mass spectrometry (nano-LC/HRMS) after immunoaffinity extraction with the objective of controlling the abuse of rhRLX-2 in horses. The limits of detection in plasma and urine are 2 pg/mL and 5 pg/mL, respectively. Two thoroughbred geldings were each administered one dose of 10 mg rhRLX-2 subcutaneously daily for 3 consecutive days. The rhRLX-2 could be detected and confirmed in the plasma and urine samples collected 105 h and 80 h, respectively, after the last dose of administration. For doping control purposes, rhRLX-2 ELISA could be used as a screening test to identify potential positive samples for further investigation using the nano-LC/HRMS methods.

Hepatitis due to Reactivation of Hepatitis B Virus in Endemic Areas Among Patients With Hepatitis C Treated With Direct-acting Antiviral Agents.

Hepatitis due to reactivation of hepatitis B virus (HBV) has been reported in patients treated with direct-acting antiviral (DAA) agents for chronic hepatitis C virus infection. We performed an observational study to determine the incidence of and factors associated with hepatitis in 327 patients receiving pan-oral DAA agents for HCV infections in areas endemic for HBV in China. Ten patients were positive for hepatitis B surface antigen (HBsAg), and 124 patients had occult HBV infection. HBV reactivation was determined by measuring HBV DNA and HBsAg status in serial serum samples collected every 2 weeks during DAA treatment and then every 4 weeks after treatment until week 12. In the total study population, 10 patients (3.1%) had hepatitis; 3 cases were associated with HBV reactivation (1 case not in the icteric phase, 1 case in the icteric phase, and 1 case with liver failure) and 7 from other causes. Testing positive for HBsAg before DAA treatment was a strong risk factor for developing hepatitis during treatment (hazard ratio, 15.0; P < .001).

Identification of porcine relaxin in plasma by liquid chromatography-high resolution mass spectrometry.

Relaxin (RLX) has demonstrated diverse pharmacological effects in various scientific and clinical studies. The emergence of porcine relaxin (pRLX) has raised concerns on the doping potential of pRLX. There have also been speculations in the horseracing industry on its covert use. To control the abuse of pRLX in equine sports, a method to detect pRLX effectively and to provide its unequivocal identification in equine biological samples is required. This paper reports on the detection and confirmation of pRLX in equine plasma by liquid chromatography-high resolution mass spectrometry. pRLX was isolated from equine plasma by immunoaffinity purification using anti-pRLX antibody-coated magnetic beads. Anti-pRLX antibody was generated in-house by purifying antisera from rabbits immunized with pRLX. The isolated pRLX was subjected to reduction of their disulfide bonds to obtain their respective A- and B-chains. The extracts were then further purified and concentrated prior to reversed-phase LC separation and high resolution accurate mass measurement. As detection of the A-chains was far more sensitive than that of the B-chains, the A-chain of pRLX was set as the targets for detection and confirmation. The limit of detection for pRLX was around 0.005 ng/mL (~ 0.86 fM) and the limit of confirmation was around 0.02 ng/mL (~ 3.4 fM). It was observed that method sensitivity was improved at least 5-fold by using an EASY-spray column and emitter in place of the conventional ESI source. The applicability of this method was demonstrated by the identification of pRLX and its metabolites in equine plasma obtained after subcutaneous administration. To our knowledge, this is the first report of a validated mass spectrometry method for the unequivocal confirmation of pRLX in any biological fluid. Copyright © 2016 John Wiley & Sons, Ltd.

Doping control analysis of anabolic steroids in equine urine by gas chromatography-tandem mass spectrometry.

Anabolic steroids are banned substances in equine sports. Gas chromatography-mass spectrometry (GC-MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography-mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC-MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast-scanning gas-chromatography-triple-quadrupole mass spectrometry (GC-MS/MS) has provided a better alternative with a significant reduction in chemical noise by means of selective reaction monitoring. Herein, we present a sensitive and selective method for the screening of over 50 anabolic steroids in equine urine using gas chromatography-tandem mass spectrometry (GC-MS/MS). Copyright © 2016 John Wiley & Sons, Ltd.

Efficacy and safety of 3-week response-guided triple direct-acting antiviral therapy for chronic hepatitis C infection: a phase 2, open-label, proof-of-concept study.

BACKGROUND: To shorten the course of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, we examined the antiviral efficacy and safety of 3 weeks of response-guided therapy with an NS3 protease inhibitor and dual NS5A inhibitor-NS5B nucleotide analogue. METHODS: In this open-label, phase 2a, single centre study, Chinese patients with chronic HCV genotype 1b infection without cirrhosis were randomly allocated by a computer program to one of three treatment groups (sofosbuvir, ledipasvir, and asunaprevir; sofosbuvir, daclatasvir, and simeprevir; or sofosbuvir, daclatasvir, and asunaprevir) until six patients in each group (1:1:1) achieved an ultrarapid virological response (plasma HCV RNA <500 IU/mL by day 2, measured by COBAS TaqMan HCV test, version 2.0). Patients with an ultrarapid virological response received 3 weeks of therapy. Patients who did not achieve an ultrarapid response were switched to sofosbuvir and ledipasvir for either 8 weeks or 12 weeks. The primary endpoint was the proportion of patients with a sustained virological response at 12 weeks (SVR12) after treatment completion, analysed in the intention-to-treat population. All patients who achieved an ultrarapid virological response were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT02470858. FINDINGS: Between April 5, 2015, and April 15, 2015, 26 eligible patients were recruited. 12 patients were assigned to sofosbuvir, ledipasvir, and asunaprevir; six to sofosbuvir, daclatasvir, and simeprevir; and eight to sofosbuvir, daclatasvir, and asunaprevir. Six patients in each group achieved an ultrarapid virological response (18 [69%]). All patients with an ultrarapid virological response who were given 3 weeks of triple therapy achieved SVR12. The most common adverse events were fatigue (one [17%] of six patients receiving sofosbuvir, ledipasvir, and asunaprevir; one [17%] of six patients receiving sofosbuvir, daclatasvir, and simeprevir; and two [33%] of six patients receiving sofosbuvir, daclatasvir, and asunaprevir) and headache (one [17%] patient in each group). No patients experienced any serious adverse events. INTERPRETATION: In this proof-of-concept study, all patients with chronic HCV without cirrhosis who achieved an ultrarapid virological response on triple direct-acting antiviral regimens by day 2 and received 3 weeks of treatment were cured, with excellent tolerability. By shortening the duration of therapy from the currently recommended 12 weeks to 3 weeks, we could drastically reduce the cost of therapy and the rate of adverse events. Further large-scale studies should be done to confirm our findings. FUNDING: Center for AIDS Research, National Institutes of Health, US Department of Energy, National Center for Research Resources and the Office of Research Infrastructure Programs, Cheng Si-Yuan (China-International) Hepatitis Research Foundation, and Humanity and Health Medical Group.

Will Sofosbuvir/Ledipasvir (Harvoni) Be Cost-Effective and Affordable for Chinese Patients Infected with Hepatitis C Virus? An Economic Analysis Using Real-World Data.

BACKGROUND: Little is known on the cost-effectiveness of novel regimens for hepatitis C virus (HCV) compared with standard-of-care with pegylated interferon (pegIFN) and ribavirin (RBV) therapy in developing countries. We evaluated cost-effectiveness of sofosbuvir/ledipasvir for 12 weeks compared with a 48-week pegIFN-RBV regimen in Chinese patients with genotype 1b HCV infection by economic regions. METHODS: A decision analytic Markov model was developed to estimate quality-adjusted-life-years, lifetime cost of HCV infection and incremental cost-effectiveness ratios (ICERs). SVR rates and direct medical costs were obtained from real-world data. Parameter uncertainty was assessed by one-way and probabilistic sensitivity analyses. Threshold analysis was conducted to estimate the price which can make the regimen cost-effective and affordable. RESULTS: Sofosbuvir/ledipasvir was cost-effective in treatment-experienced patients with an ICER of US$21,612. It varied by economic regions. The probability of cost-effectiveness was 18% and 47% for treatment-naive and experienced patients, and it ranged from 15% in treatment-naïve patients in Central-China to 64% in treatment-experienced patients in Eastern-China. The price of 12-week sofosbuvir/ledipasvir treatment needs to be reduced by at least 81% to US$18,185 to make the regimen cost-effective in all patients at WTP of one time GDP per capita. The price has to be US$105 to make the regimen affordable in average patients in China. CONCLUSION: Sofosbuvir/ledipasvir regimen is not cost-effective in most Chinese patients with genotype 1b HCV infection. The results vary by economic regions. Drug price of sofosbuvir/ledipasvir needs to be substantially reduced when entering the market in China to ensure the widest accessibility.

An evaluation of factors affecting preference for immediate, delayed or no breast reconstruction in women with high-risk breast cancer.

OBJECTIVE: Women with locally advanced breast cancer face many conflicting issues affecting their choice of immediate versus delayed versus no breast reconstruction (BR). This single-centre pilot study assessed high-risk women's reasons and priorities in choosing the timing and type of BR in a setting where all clinically feasible options were discussed with all women. METHODS: Fifty-one women from a metropolitan breast oncology practice, who were likely to require post-mastectomy radiotherapy (PMRT), were recruited after making their decision about BR. Participants completed a questionnaire (69% preoperatively), adapted from Reaby (1998), evaluating the factors affecting their decision. Responses were subsequently classified into eight issue-based domains (feeling normal, feeling good, being practical, influence of others, expectations, fear, timing and unnecessary). Demographic and clinical data were also collected. RESULTS: There were 32 immediate BR (IBR = 63%), seven delayed BR (DBR = 13%) and 12 no BR (NBR = 23%). Analysis using the chi square test showed women over 60 were more likely to choose NBR (p = 0.005), while women living with a partner were more likely to choose IBR (p = 0.032). The most relevant domains for both IBR and DBR were 'feeling good' and 'feeling normal'; and for NBR were 'unnecessary' and 'being practical'. Although all women understood pre-operatively the potential aesthetic limitations of PMRT, 63% still chose IBR. CONCLUSIONS: These data will enable clinicians, researchers and women with breast cancer to gain a clearer understanding of the factors that impact on the choice and timing of BR in women requiring PMRT, a major breast cancer survivorship decision. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic studies of oxyguno in horses.

Oxyguno (4-chloro-17α-methyl-17ß-hydroxy-androst-4-ene-3,11-dione) is a synthetic oral anabolic androgenic steroid commercially available without a prescription. Manufacturers of oxyguno claim that its anabolic effect in metabolic enhancement exceeds that of the classic anabolic steroid testosterone by seven times, but its androgenic side-effects are only twelve percent of testosterone. Like other anabolic androgenic steroids, oxyguno is prohibited in equine sports. The metabolism of oxyguno in either human or horse has not been reported and therefore little is known about its metabolic fate. This paper describes the in vitro and in vivo metabolic studies of oxyguno in racehorses with an objective to identify the most appropriate target metabolites for detecting oxyguno administration. In vitro studies of oxyguno were performed using horse liver microsomes. Metabolites in the incubation mixtures were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry in the EI mode after trimethylsilylation. In vitro metabolites identified include the stereoisomers of 4-chloro-17α-methyl-androst-4-ene-3-keto-11,17ß-diol (M1a & M1b); 20-hydroxy-oxyguno (M2); and 4-chloro-17α-methyl-androst-4-ene-3-keto-11,17ß,20-triol (M3). These novel metabolites were resulted from hydroxylation at C20, and/or reduction of the keto group at C11. For the in vivo studies, two geldings were each administered orally with a total dose of 210 mg oxyguno (52.5 mg twice daily for 2 days). Pre- and post-administration urine and blood samples were collected for analysis. The parent drug oxyguno was detected in both urine and blood, while numerous novel metabolites were detected in urine. The stereoisomers (M1a & M1b) observed in the in vitro studies were also detected in post-administration urine samples. Three other metabolites (M4 - M6) were detected. M4, 4-chloro-17α-methyl-androstane-11-keto-3,17ß-diol, was resulted from reductions of the olefin group at C4 and the keto group at C3. M5 was resulted from hydroxylation at C20 and two reductions at either the olefin group at C4, the keto group at C3, or the keto group at C11. M6 was assigned as the 17-epimer of oxyguno. The major biotransformation pathways of oxyguno identified were reduction, hydroxylation and epimerisation. The structures of all metabolites were tentatively assigned by mass spectral interpretation. The longest detection time observed in urine was up to 10 h for the in vivo metabolite M4. Urinary and plasma oxyguno decreased rapidly and was no longer detectable at respectively 7 and 12 h post-administration. The above studies have provided useful information for the monitoring of oxyguno administration in racehorses.

Liquid chromatography-mass spectrometry analysis of five bisphosphonates in equine urine and plasma.

Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography - tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples.

Control of the misuse of testosterone in castrated horses based on an international threshold in plasma.

Testosterone is an endogenous steroid produced primarily in the testes. Trace levels of testosterone are found in urine samples from geldings, as testosterone is also secreted by the adrenal. An international threshold of free and conjugated testosterone in urine (20 ng/mL) was adopted by the International Federation of Horseracing Authorities (IFHA) in 1996 for controlling testosterone misuse in geldings. In view of the recent popularity of using blood in doping control testing, it is necessary to establish a threshold for testosterone in gelding plasma. A liquid chromatography-mass spectrometry (LC/MS) method was developed for quantifying low levels of free testosterone in gelding plasma. Based on a population study of 152 post-race plasma samples, the mean ± SD concentration of plasma testosterone was determined to be 14.7 ± 6.8 pg/mL. Normal distribution could be obtained after square-root or cube-root transformation, resulting in respective tentative thresholds of 49 or 55 pg/mL (corresponding to a risk factor of less than 1 in 10 000). A rounded-up threshold of 100 pg/mL of free testosterone in plasma was proposed. Based on the administration of Testosterone Suspension 100 to six geldings, the same average detection time of 14 days was observed in either plasma or urine using the proposed plasma threshold and the existing international urine threshold. The maximum detection time was 18 days in plasma and 20 days in urine. The results demonstrated the proposed plasma threshold is effective in controlling the misuse of testosterone in geldings. Similar results were subsequently obtained in Europe, and this proposed threshold was adopted by IFHA in October 2013.

Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor regulating granulopoiesis. The recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used for the treatment of granulopenia in humans. Filgrastim is a rhG-CSF analogue and is marketed under various brand names, including Neupogen(®) (Amgen), Imumax(®) (Abbott Laboratories), Neukine(®) (Intas Biopharmaceuticals) and others. It is banned in both human and equine sports owing to its potential for misuse. In order to control the abuse of filgrastim in equine sports, a method to identify unequivocally its prior use in horses is required. This study describes an effective screening method for filgrastim in equine plasma by enzyme-linked immunosorbant assays (ELISA), and a follow-up confirmatory method for the unequivocal identification of filgrastim by analysing its highly specific tryptic peptide (1)MTPLGPASSLPQSFLLK(17). Filgrastim was isolated from equine plasma by immunoaffinity purification. After trypsin digestion, the mixture was analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Filgrastim could be detected and confirmed at 0.2ng/mL in equine plasma. The applicability of the ELISA screening method and the LC/MS/MS confirmation method was demonstrated by analysing post-administration plasma samples collected from horses having been co-administered with epoetin alfa as recombinant human erythropoietin (rhEPO) and filgrastim as rhG-CSF. rhEPO and filgrastim could be detected in plasma samples collected from horses for at least 57 and 101h respectively. To our knowledge, this is the first identification of filgrastim in post-administration samples from horses.

Increasing breast reconstruction rates by offering more women a choice.

BACKGROUND: Breast reconstruction (BR) following mastectomy for breast cancer is safe and has high rates of patient satisfaction, yet only around 12% of Australian women undergo BR. This study presents BR rates and outcomes from a specialist practice that discusses reconstruction options with all women medically suitable for BR. METHODS: Retrospective clinical study of all women that had undergone therapeutic mastectomy between 2009 and 2011. Patient, tumour and adjuvant therapy factors, and surgical complication rates, were compared between BR and no BR (NBR) patients. RESULTS: Of the 331 women who had mastectomy for cancer, 136 (41%) had BR, with the vast majority (132, 97%) opting for immediate BR (IBR). Factors significantly associated with BR were young age, pure ductal carcinoma in-situ (DCIS), menopausal status and private health insurance. The main reasons for NBR were patient choice (88/195, 45%) and surgeon's perception of high-risk tumours (63/195, 32%). At mean follow-up of 15.6 months, five patients had developed local or distant recurrence (2 BR, 3 NBR). IBR did not cause significant delays in commencement of adjuvant therapy, and the BR group had a lower rate of surgical complications. DISCUSSION: A BR rate of 41%, over three times the national average, was achieved when BR was discussed with all patients. This significant gain in BR rate was not accompanied by a commensurate increase in adverse outcomes, providing evidence that expanding the indications for BR to women who were previously not considered eligible is a valid option.