Vitamin D has received significant attention from clinical societies, researchers, and the general population in recent years. While 25-hydroxyvitamin D (25(OH)D) is the most commonly-used biomarker of vitamin D status, 1α,25-dihydroxyvitamin D (1,25(OH)₂D), its bioactive form, plays a critical role in regulating calcium and phosphorus homeostasis and is also involved in the immune system and cellular differentiation. Consequently, accurate measurements of 1,25(OH)₂D can aid in the differential diagnosis of calcium-related disorders such as hypocalcemia in vitamin D-dependent rickets and hypercalcemia due to inappropriate increase of serum 1,25(OH)₂D in granulomatous diseases. However, due to its lipophilicity and very low circulating concentration, the measurement of 1,25(OH)₂D is particularly challenging. Over the past several decades, numerous efforts have been made to develop sensitive, specific, and practical laboratory methods for measuring 1,25(OH)₂D. Methods using radioreceptor assay, radioimmunoassay, enzyme immunoassay, enzyme-linked immunosorbent assay, automated chemiluminescent immunoassay, and liquid chromatography-tandem mass spectrometry have been described. Each of these methods has unique advantages and limitations, and some are no longer used. Despite the sophisticated methods in use today, substantial variations between methods still exist. A concerted effort toward standardization of 1,25(OH)₂D measurement is needed to ensure accurate and reliable results across laboratories and methods.

Purpose: Bladder outlet obstruction induces detrusor hypertrophy and it can eventually lead to decreased bladder smooth muscle contractility. Heat shock protein 20 is the proposed mediator of force suppression in vascular smooth muscle. We investigated whether heat shock protein 20 could also mediate the decreased contractility observed in partially obstructed rat bladders. Materials and Methods: Female Wistar rats (Harlan Laboratories, Indianapolis, Indiana) were randomized to partial urethral ligation or sham ligation. After 3 weeks the rats were sacrificed, and the bladders were harvested, frozen, homogenized and analyzed for heat shock protein 20 content by Western blot immunoreactivity. The content of myosin regulatory light chain, a constitutively expressed protein, was determined as a control. Bladder smooth muscle strips were dissected from some rats and mounted for force generation measurement. Results: At cystectomy obstructed bladders were significantly heavier and had more residual urine compared to sham operated bladders. Heat shock protein 20 immunoreactivity was significantly increased a mean ± 1 SEM of 1.9 ± 0.3-fold in obstructed vs sham operated bladders. Control protein myosin regulatory light chain immunoreactivity did not significantly differ in obstructed and sham operated bladders. Maximal stress, that is force per cross-sectional area, was significantly decreased in obstructed vs sham operated bladders. Human bladder was found to express immunoreactive heat shock protein 20. Conclusions: We noted that partially obstructed rat bladders 1) express higher levels of heat shock protein 20 and 2) generate less stress than sham operated bladders. These data suggest the possibility that heat shock protein 20 over expression could at least partially mediate the decreased contractile activity observed with partial bladder outlet obstruction. The mechanism for increased heat shock protein 20 expression is unknown but it may involve increased mechanical stress or hypoxia from urethral obstruction. Human bladder expressed immunoreactive heat shock protein 20, suggesting that a similar mechanism could potentially occur in humans. If confirmed in humans, patients with clinical conditions that result in detrusor hypocontractility could potentially benefit from pharmacological interventions aimed at inhibiting heat shock protein 20.

As an active metabolite of a commonly prescribed immunosuppressant, mycophenolic acid (MPA) levels are often monitored to prevent organ rejection following a transplant. Triazoles are often prescribed for treatment of invasive fungal infections in immunocompromised patients. Due to the variability in individual pharmacokinetics and drug-drug interactions, therapeutic drug monitoring is recommended for triazole antifungals. A multiplex LC-MS/MS assay has been developed that can quantify both MPA and triazole drugs in serum. A sample preparation procedure was established to spike in internal standard compounds and precipitate proteins. Reversed-phase chromatographic separation was performed on a C18 column with an analysis time of five minutes per sample. The mass spectrometer was operated in multiple reaction monitoring mode. The method was validated on two HPLC systems interfaced with either a Triple Quad 6500 or an API 4000 instrument. The multiplex assay was linear over a wide dynamic range with analyte measurable ranges of 0.4–48 μg/mL for MPA, 0.1–12 μg/mL for posaconazole, and 0.2–24 μg/mL for voriconazole, itraconazole, hydroxyitraconazole, and isavuconazole. The between-day and intraday imprecisions were less than 10 %. Limits of detection were below 0.04 ug/mL with limits of quantitation below 0.2 μg/mL. Method comparison studies against the current in-house method met acceptance criteria. The instrument comparison study demonstrated a strong correlation between data collected from the two systems. A robust multiplex LC-MS/MS assay was developed and validated for monitoring MPA and triazoles drug levels in a clinical laboratory. The assay performance on two distinct instruments was acceptable and comparable.

Background: Testosterone is an androgenic hormone that plays important roles in both males and females. The circulating levels of total testosterone vary from 1 to 1480 ng/dL. High-throughput immunoassays often lack accuracy in lower concentration ranges (below 100 ng/dL), particularly when used for females or children. To address this limitation, we developed a total testosterone LC-MS/MS assay on three instruments. Methods: Sample preparation began with the dilution and conditioning of 200 µL of serum. A supported liquid extraction cartridge was used to extract the analyte from biological matrices. Chromatographic separation was achieved using a C18 column with a runtime of 5 min per sample. This assay was validated on a Triple Quad 6500 and an API 4500 instrument. Results: Method validation was carried out according to the CLSI C62-ED2 guideline and our hospital protocol. The within-day coefficient of variation (CV) was less than 10% and the between-day CV was less than 15%. The assay had a limit of quantitation of 0.5 ng/dL with an analyte measure range of 2–1200 ng/dL. A comparison using Deming regression and Bland–Altman plots showed that this assay correlated well with a reference method. The results from the API 4500 and an Orbitrap were consistent with those from the TQ 6500. Both serum-separator tubes (BD) and serum-activator tubes were found to be suitable. Conclusions: We successfully developed and validated a robust total testosterone LC-MS/MS assay for routine clinical testing. This assay was harmonized across two triple quadrupole instruments and one high-resolution mass spectrometer.