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The Tirimbina Biological Reserve is located in the lowlands on the Atlantic versant of Costa Rica. This study provides an updated comprehensive herpetofauna species list and summarizes the results of all the herpetofauna research conducted at Tirimbina over the last ten years (2009–2019) across various microhabitats, both daytime and nighttime. We also added historical records from occasional sightings and reports from researchers, staff, visitors, interns, fellows, and volunteers since the 1990’s. We found 52 amphibian and 78 reptile species on the reserve, including a few species considered at–risk according to the IUCN Red List. We conclude that Tirimbina is a herpetofauna biodiversity hot spot in Costa Rica because it provides unique habitat characteristics for a variety of species, including habitat for both forest and forest–edge specialists. Tirimbina Biological Reserve for the Tirimbina–UCR research grant. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biodiversidad y Ecología Tropical (CIBET) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biología

Background: The specificity of screening for congenital adrenal hyperplasia by direct measurement of 17-hydroxyprogesterone in filter paper dried blood spot samples by immunoassay is low and has a high false-positive rate. In order to reduce the false-positive rate of this test, we developed a rapid, robust, specific confirmatory procedure in which cortisol, 4-androstene-3,17-dione and 17-hydroxyprogesterone were measured simultaneously by ultra-performance liquid chromatography-tandem mass spectrometry. Methods: After extraction, samples were analysed by ultra-performance liquid chromatography-tandem mass spectrometry and 17-hydroxyprogesterone was quantified accurately. Other steroids were determined using stable deuterated internal standards. In total, 25 patient blood spot samples and 92 control samples were analysed. Results: The assay was linear for 17-hydroxyprogesterone, with a coefficient of determination >0.997 and imprecision ≤6.5%. An upper limit of normal for 17-hydroxyprogester-one of 4.45 nmol/L was established by analysing a cohort of samples from unaffected newborns. In addition, a cut-off of 3.5 for the peak areas ratio (17-hydroxyprogesterone+4-androstene-3,17-dione)/cortisol, allows confirmation of the affected steroidogenic enzyme. Conclusions: A high throughput method for the detection of steroids related to congenital adrenal hyperplasia has been developed, allowing the false-positive rate associated with screening for 17-hydroxyprogesterone by immunoassay to be determined.

Background: The measurement of urinary free cortisol (UFC) is commonly used in the investigation of possible Cushing's syndrome. With the recent availability of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in hospital laboratories, we wanted to develop a specific UFC LC-MS/MS method and compare it with our current immunoassay method and develop a new LC-MS/MS reference range if required. Methods: A UFC LC-MS/MS method using deuterated cortisol as an internal standard was optimized using solid-phase extraction as a clean-up procedure. The multiple reaction-monitoring transitions used for the detection of cortisol and deuterated cortisol were 363.1>121 and 365.1>121.8, respectively. The method was investigated regarding precision, linearity, sensitivity, recovery and interference. UFC was measured by the in-house urine adapted ACS:180 serum cortisol immunoassay and the developed LC-MS/MS method in 110 urine samples from patients being investigated for possible Cushing's syndrome. Results: The within-batch precisions ( n=25) of the LC-MS/MS method were 7.6%, 4.5% and 3.3% at 25.0 nmol/L, 49.6 nmol/L and 344.6 nmol/L, respectively; the between-batch precisions ( n = 10) were 9.4%, 9.4% and 8.4%, respectively, at these concentrations. The method is sensitive down to 5 nmol/L and linear up to at least 1000 nmol/L. The method showed adequate cortisol recovery and no interference from the numerous drugs and steroids tested. The total run time for 20 samples, including sample preparation, was 120 min. A scatter plot of paired UFC measurements on the LC-MS/MS and the ACS:180 gave the equation: LC-MS/MS=0.408 (ACS:180)+2.65, r2 = 0.6664. The 24-h measured UFC results on 110 samples (25 men and 85 women) were positively skewed. After log transformation the data were less skewed, and following back transformation of the lower 97.5th centile, the upper limit of normal was 165 nmol/24 h. The 95th centile of the untransformed data was 146 nmol/24 h ( n=110, 25 men and 85 women). Separated by sex, the 95th centile was 152 nmol/24 h for men ( n=25) and 141 nmol/24 h for women ( n=85). Conclusions: We have developed a UFC LC-MS/MS method with a solid-phase extraction clean-up step. The method shows adequate performance and is suitable for routine laboratory use. The mixed sex ( n=110, men=25, women=85) reference range was up to 165 nmol/24 h or 146 nmol/24 h, depending on how the data are manipulated.

Background Internal standards are essential in quantitative mass spectrometry (MS) assays to correct for variability in sample extraction and ionization at the source. In liquid chromatography MS assays, analogues of the analyte with several atoms replaced by their stable isotopes, e.g. 2H (D, deuterium) are often used as internal standards. Methods Possible interference by naturally occurring isotopes of an analyte in the internal standard channel in a liquid chromatography tandem MS assay was assessed using cortisol and its deuterated internal standard, D2-cortisol, as an example. Mass spectra were analysed and standard curves were prepared with varying concentrations of internal standard to determine the extent of any interference. Results The mass spectra showed that a naturally occurring isotope of cortisol at m/z 365 acts in the same way as D2-cortisol and fragments to give a daughter ion of the same m/z. Cortisol-365 can therefore falsely, but significantly, increase the amount of internal standard detected, and this will concomitantly decrease the relative response for cortisol. The standard curves with varying concentrations of internal standard showed that this phenomenon can affect the linearity of an assay. Conclusions Our results show that care is needed in assay development when doubly deuterated internal standards are used. Interference by naturally occurring isotopes of the analyte of interest in the internal standard transition is possible and it is important to ensure that an appropriate internal standard concentration is chosen that permits linearity of the assay over the required range.

Background Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed. Methods The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturer's instructions. Results When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were 2 = 0.9937). Conclusions The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.

Background: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. Methods: We used stable-isotope dilution liquid chromatography–tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 μL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. Results: Intra- and interassay imprecision was 8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. Conclusions: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Prednisolone is a commonly prescribed corticosteroid used in the treatment of many diseases. Despite high doses of prednisolone, some patients appear to have subtherapeutic concentrations of the drug. It would be useful to measure prednisolone in this group to determine if they have poor absorption or compliance. Hence, we have developed a liquid chromatography-tandem mass spectrometry method for the determination of prednisolone in serum. Chromatography was performed using a C18 column, giving a retention time for both prednisolone and deuterated prednisolone (internal standard) of 1.6 min. Two transitions were monitored for both prednisolone and deuterated prednisolone. These were m/z 361.2>343.0 and m/z 361.2>146.9 for prednisolone, and m/z 367.2>349.0 and m/z 367.2>149.9 for the internal standard. The intra- and inter-batch imprecision was

Background: Current sampling for total homocysteine (tHcy) is problematic, requiring plasma separation within 15 min. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the measurement of tHcy in plasma and dried blood spots and to determine whether the dried blood spot concentration could be used to predict plasma concentrations of tHcy. Methods: LC-MS/MS methodology was optimized to measure tHcy in plasma and dried blood spots. Fifty blood samples collected from heart transplant patients were used to form dried blood spots and for plasma analysis. Plasma tHcy was also measured using the Abbott IMx1 method and values were compared to the tHcy concentrations determined in plasma and dried blood spots using LC-MS/MS methodology. Results: The plasma tHcy LC-MS/MS results compared well with the IMx values: LC-MS/MS=1·18(IMx)-0·44 ( r2=0·915). The within-batch precision ( n =10) of the plasma LC-MS/MS method was < 2·0% at 14·6 and 37·7 µmol/L, respectively; the between-batch precision ( n=10) was 5·0 and 8·0%, respectively, at these concentrations. The method was found to be sensitive down to 1 µmol/L and linear up to at least 100 µmol/L. Dried blood spot LC-MS/MS results were considerably lower than the plasma IMx values ( P < 0·0001): dried blood spot LC-MS/MS=0·33IMx+1·77 ( r2=0·682). The within-batch precision ( n=20) of the dried blood spot LC-MS/MS method was 7·3% and 4·7% at concentrations of 4·0 and 7·9 µmol/L, respectively; the between-batch precision was 12·6% and 7·9% at concentrations of 5·1 and 8·0 µmol/L, respectively. To assess whether dried blood spots are suitable as a screening test to predict plasma tHcy concentrations, arbitary cut-off levels were compared. If it is assumed that a plasma tHcy concentration of >15 µmol/L is raised, a dried blood spot result of >6·8 µmol/L has a sensitivity and specificity in detecting a raised plasma tHcy of 83·3% and 96·2%, respectively, and a positive and negative predictive value of 95% and 86%, respectively, with an efficiency of 90%. Use of a dried blood spot cut-off concentration of 6·2 µmol/L for predicting high plasma tHcy concentrations (above 15 µmol/L) has a sensitivity and specificity of 95·8% and 73·1%, respectively, positive and negative predictive values of 76% and 95%, respectively, and an efficiency of 84%. Conclusions: We have developed a precise and accurate LC-MS/MS method for measuring plasma tHcy concentrations, which uses a small volume of plasma and is suitable for routine use. A satisfactory LC-MS/MS method for the measurement of tHcy in dried blood spots was also developed; this method might be useful in routine screening for raised plasma concentrations of tHcy.