Unstripped Serum Research Articles

Simultaneous determination of levothyroxine and its metabolite liothyronine in human serum by using LC-ESI-MS/MS and its bioequivalence application

Levothyroxine is a synthetic thyroid hormone, used to treat hypothyroidism and related conditions. It has been a widely used and effective medication for many decades. The bioanalytical method is developed for the determination of endogenously present levothyroxine and its metabolite, liothyronine, in both stripped and un-stripped serum, using liquid chromatography-tandem mass spectrometry. Determining an endogenously present analyte is indeed a critical step in pharmacokinetic studies and determining the therapeutic dose of a drug. The term “stripped” typically refers to the removal of endogenous analytes that interfere with the accurate quantification of the analyte of interest. Method employed with, Kinetex® polar C18 (100 × 4.6 mm 2.6 μm) analytical column for chromatographic resolution by using, pump A as acetic acid in water and pump B as acetic acid in methanol. The mass spectrometer used with triple quadrupole operating in the positive ion mode, specifically using multiple reaction monitoring. Multiple reaction monitorin is a highly delicate and discriminating technique for quantifying specific analytes. The transitions are as follows: analyte transition: 777.55→731.65, metabolite transition: 651.70→605.85, IS analyteTransition: 780.60→734.75, and IS metaboliteTransition: 661.60→614.65. A resolution factor of 0.5 was achieved for levothyroxine and liothyronine. Linearity for levothyroxine in a range of 20.0–1000 ng/ml and liothyronine in a range of 0.3–15.0 ng/ml was employed. Method validation was performed to conduct stability studies and demonstrate the reproducibility for accuracy and reliability for LC-ESI-MS/MS assay. The proposed bioanalytical method is highly sensitive and selective, has a minimal processing volume, faster run time, no matrix impact, and the ability to measure analytes in different types of serum (stripped and normal). The method was valuable for both research and clinical laboratories and can provide reliable results.

Evaluation of a one-step sample preparation protocol for analysis of total serum testosterone by LC–MS/MS

In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. The difference in accuracy was less than 5% for adult sera, while it was less than 10% for prepubescent sera. To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. The baseline comparison of LC–MS/MS chromatograms of samples indicated that samples prepared by the one-step procedure were of the same quality as those prepared by the two-step procedure; however, recovery in unaltered serum using the one-step procedure was approximately 15% greater across the low to high concentration range. Furthermore, recovery using the one-step procedure was more consistent between stripped and unstripped serum. Lipids were removed efficiently in the two-step procedure as verified by monitoring the typical phospholipid MRM transition of m/z 496 → 184. For every sample processed by the one-step procedure, a one-minute online column wash with 95% methanol was able to remove 95% of the bound lipids, thereby providing a column life-time approximately equivalent to that for the two-step procedure. The presented data indicate that the one-step procedure could replace the two-step procedure while maintaining accuracy, saving time, increasing recovery, and minimizing the potential for errors with the fewer steps required.

Quantification of dehydroepiandrosterone in human serum on a routine basis: development and validation of a tandem mass spectrometry method based on a surrogate analyte.

In the clinical laboratories, dehydroepiandrostenedione (DHEA) is usually quantified by immunoassay-based methods, which are often affected by cross-reactivity with endogenous interferences, such as 4-androsten-3β-ol-17-one. The interfering compounds lead to a poor accuracy of the measurements, mainly at a low concentration level. The present paper describes a validated method based on tandem mass spectrometry coupled to liquid chromatography, for the accurate quantification of DHEA in serum. The peculiarity of this method is the use of calibrators and quality controls prepared by adding measured amounts of DHEA-D5, a stable isotope-labeled analogue of DHEA, to real serum from healthy subjects. DHEA-D5 is used in place of DHEA, which is usually present in unstripped serum at physiological levels, as it has the same basic structure, provides an equivalent instrumental response, and can be easily distinguish by DHEA by mass spectrometry due to its different m/z value. The method proved to be sensitive, with a LLOD of 0.09ng/mL and a LLOQ of 0.23ng/mL, and selective, with overall performances that allow its use on a routine basis.

The conversion of 16β hydroxyldehydroepiandrosterone in human serum

The circulating levels of 16β hydroxydehydroepiandrosterone (16β OH-DHEA) are at the limit of detection (less than 10pg/mL), unlike the serum concentrations of 16α-hydroxydehydroepiandrosterone (16α OH-DHEA, 10–300pg/mL) in premenopausal, postmenopausal and male serum. A major reason could be the rapid conversion of 16β OH-DHEA to 5-androstene-3beta, 17beta-diol 16 one (3β, 17β-diol 16-oxo) in serum due to the stereospecific structure of 16β OH-DHEA. In ultrapure H2O, there is no apparent conversion observed while 16β OH-DHEA (10ng/mL) spiked in stripped or unstripped serum is quickly converted to 3β, 17β-diol 16-oxo at room temperature. During this conversion, a further converted product was observed with a difference in molecular weight of 16Da from that of 16β OH-DHEA and 3β, 17β-diol 16-oxo, which could be their hydroxylation product, i.e. triol-ketone. Under basic conditions, further conversion occurs. The present data can explain the practically undetectable concentration of serum 16β OH-DHEA while 3β, 17β-diol 16-oxo is at the level of less than 50pg/mL. Serum concentrations of (0.0–9.9pg/mL for 16β OH-DHEA, 8.9–50.7pg/mL for 3β, 17β-diol 16-oxo and 10.0–285.0pg/mL for 16α OH-DHEA are measured in sera of premenopausal, postmenopausal women and men over 50years of age.

A rapid and sensitive UPLC–MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women

Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC–MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women’s serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200μL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.

Validated LC–MS/MS simultaneous assay of five sex steroid/neurosteroid-related sulfates in human serum

Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno‑based assays before the use of liquid-chromatography tandem mass spectrometry (LC–MS/MS). In the present study, a validated LC–MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1‑S), androsterone sulfate (ADT‑S), pregnenolone sulfate (Preg‑S) and allopregnanolone sulfate (Allopreg‑S). E1‑S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to −10 to −15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1‑S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40ng/mL, 40pg/mL, 5ng/mL, 1.5ng/mL and 0.25ng/mL for DHEA‑S, E1-S, ADT‑S, Preg‑S and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1‑S, ADT‑S and Preg‑S while for Allopreg‑S, the accuracy is within the 15% range. The interday coefficient variance is 5.5–9.5% for the low limits of quantification of all five compounds while values of 1.3–9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines.

A sensitive, simple and robust LC–MS/MS method for the simultaneous quantification of seven androgen- and estrogen-related steroids in postmenopausal serum

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC–MS or GC–MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC–MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid–liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1pg/mL, 4pg/mL, 50pg/mL, 10pg/mL, 100pg/mL, 500pg/mL and 100pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4pg/mL with S/N≥5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1–84.7% for E2, 83.6–94.5% for E1, 88.2–90.3% for Testo, 82.0–90.6% for DHT, 84.9–92.0% for 5-diol, 88.1–93.8% for DHEA and 86.2–90.3% for 4-dione, respectively. A good linearity is obtained with R>0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1–8.9% for the low limits of quantification of all seven compounds with 0.7–6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC–MS/MS and LC–MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.

PO9-216 AGE-DEPENDENT EFFECT ON RESISTIN BLOOD LEVEL AND ITS ROLE IN THE INFLAMMATION AND CALCIFYING PROCESS OF THE AORTIC VALVE

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC–MS or GC–MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC–MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid–liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1 pg/mL, 4 pg/mL, 50 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL and 100 pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4 pg/mL with S/N ≥ 5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1–84.7% for E2, 83.6–94.5% for E1, 88.2–90.3% for Testo, 82.0–90.6% for DHT, 84.9–92.0% for 5-diol, 88.1–93.8% for DHEA and 86.2–90.3% for 4-dione, respectively. A good linearity is obtained with R > 0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1–8.9% for the low limits of quantification of all seven compounds with 0.7–6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC–MS/MS and LC–MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.

Candida albicans morphogenesis is influenced by estrogen.

Conversion of Candida albicans from yeast to mycelial growth is believed to be associated with the organism's virulence. We investigated the role of mammalian hormones in initiating this transformation. Three clinical isolates of Candida albicans were tested for their ability to produce germ tubes under various conditions. Controlled hormonal conditions were provided by stripping rabbit serum with activated charcoal. Steroid compounds under investigation were added back to the stripped serum and yeast were inoculated into the test materials. Microscopic counts of germinated versus ungerminated cells were used as an indicator of morphogenic transformation. The percent of yeast cells germinating was profoundly reduced in stripped compared to unstripped serum. The addition of 1 microM estradiol, cholesterol or testosterone only slightly increased levels of germination above that seen in controls. Estradiol at concentrations 100 times less, however, proved a strong inducer of germination. Cholesterol did not synergize germination when combined with estradiol and the alpha isomer of estradiol had almost no activity as an inducer of morphogenic change in Candida albicans. We conclude that beta estradiol was a morphogenic inducer in three clinical isolates of Candida albicans but only at concentrations typical in vivo.