Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber

Abstract

RATIONALE: Analysis of steroids from precious blubber biopsies obtained from marine mammals, especially endangered species, can provide valuable information on their endocrine status. Challenges with currently used ELISA methodology include lack of absolute quantitation and incompatibility with multiple steroids analysis due to limited biopsy mass. Development of a sensitive, accurate analytical method for this purpose is critical. METHODS: A nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) method was validated for sensitive, specific and quantitative analysis of three steroid hormones, without derivatization, extracted from 50 mg blubber samples. Data was acquired with an LTQ XL ion trap mass spectrometer in positive ion mode, using single reaction monitoring. All three steroids were analyzed in a single run. Cholic acid was used as a surrogate internal standard for quantitation due to its steroidal structure and lack of measurable endogenous levels in blubber. RESULTS: The lowest limits of quantitation for progesterone, testosterone, and hydrocortisone were significantly improved compared to previous studies using conventional LC/MS/MS. The lowest limit of detection was 7 fg/μL using a 1 μL injection volume. Calibration curves for steroid quantification showed good linearity (r 2 >0.99) between 14 and 3620 fg/μL, and accuracy was <20% for interday and <10% for intraday. After validation, the method was successfully applied to quantification of steroids in gray whale blubber samples. CONCLUSIONS: The nanoLC/MS/MS method is more sensitive than traditional LC/MS/MS for steroid analysis. It is also compatible with other important biopsy analyses due to its small blubber mass requirement. This will benefit the reproductive and stress assessments for all marine mammals, particularly endangered populations.

A novel method to measure steroid hormone concentrations in walrus bone from archaeological, historical, and modern time periods using liquid chromatography/tandem mass spectrometry

“…The accuracy of our method for extracting steroid hormones from bone ranged from 93–111%, all within acceptable values for measuring steroid hormones via LC/MS/MS (Table , 83.5–115.4% from Zhang et al). In addition, these accuracy values are similar to studies that measured progesterone, testosterone, and hydrocortisone in gray whale blubber (88–118%), and progesterone, testosterone, and cortisol in bottlenose dolphin blubber (84–112%) via LC/MS/MS.…”

“…Where expected concentration (EC) is divided by mean actual concentration measured in spiked bone tissue (MAC) and then multiplied by 100 (Table ) . Steroid hormone concentrations in marine mammal serum and blubber have been previously validated using LC/MS/MS methods …”

“…However, immunoassays require relatively large sample masses and multiple assays for multi‐hormone analyses, which increases required lab time . In addition, cross‐reactivity with target steroid hormone metabolites can lead to inflated hormone concentrations . Furthermore, due to complicated logistics of collecting tissue samples from free‐ranging marine mammals and animal care standards for managed populations, marine mammal biopsies, blow samples, fecal samples, etc., once obtained, are relatively small and are slated for multiple different analyses, (e.g., contaminants, fatty acids, disease) .…”

“…Furthermore, due to complicated logistics of collecting tissue samples from free‐ranging marine mammals and animal care standards for managed populations, marine mammal biopsies, blow samples, fecal samples, etc., once obtained, are relatively small and are slated for multiple different analyses, (e.g., contaminants, fatty acids, disease) . Thus, researchers need to efficiently analyze tissue samples and have been transitioning from using immunoassays to more sophisticated analyses, like liquid chromatography/tandem mass spectrometry (LC/MS/MS) …”

“…LC/MS/MS measures the amount of the actual target analyte, resulting in low cross‐reactivity of metabolites and greater accuracy of actual steroid hormone concentrations in samples . Recently, a variety of LC/MS/MS methods have been developed to measure multiple hormones in a single sample of marine mammal blubber, whale blow, and serum . Serum represents circulating hormone concentrations (short‐term), while blubber represents approximately weekly to monthly hormone concentrations .…”