Progesterone In Human Serum Research Articles

Quantitative of progesterone using isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry

A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum was reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. In the study, the recovered progesterone concentration determined by the assay showed good robustness and constancy in comparison to conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.Advantages of this technique includes:• This study describes a MALDI-TOF/MS method for the determination of serum progesterone.• The technique is simple and easy to apply on MALDI-TOF/MS for serum progesterone analysis.• The correlation coefficient between MALDI-TOF MS and RIA was 0.981 for serum progesterone.

Quantitative analysis of progesterone using isotope dilution-matrix-assisted laser desorption ionization-time of flight mass spectrometry as a reference procedure for radioimmunoassay

BackgroundProgesterone is one of the female steroid hormones and plays an important role in the menstrual cycle and during pregnancy. It is especially important in preparing the uterus for the implantation of the blastocyst and maintaining pregnancy. The concentration in human serum is measured to determine the ovarian function retroactively and the cause of abortion in early pregnancy. MethodsA quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum is reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. ResultsProgesterone and the internal standard, 13C3-progesterone, were measured in the selected reaction monitoring mode for the transitions m/z 315.4 to 108.9 and m/z 318.4 to 111.9, respectively. We calculated the peak area ratio of progesterone to 13C3-progesterone. The progesterone concentration in human serum was calculated by substituting the peak area ratio into an isotope dilution calibration curve, and then compared with the radioimmunoassay. ConclusionsIn the study, the concentrations of serum progesterone were measured, and the recovered progesterone concentration determined by the assay showed good robustness and consistency in comparison to the conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone.

Performance enhancement in the measurement of 5 endogenous steroids by LC–MS/MS combined with differential ion mobility spectrometry

BackgroundChallenges for steroid analysis by LC–MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. Methods250μL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC–DMS–MS/MS. The method was validated and compared with immunoassays and LC–MS/MS methods of reference laboratories. ResultsInter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r2) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively. ConclusionsWe developed and validated an LC–DMS–MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.

Measurement of progesterone in human serum by isotope dilution liquid chromatography–tandem mass spectrometry and comparison with the commercial chemiluminescence immunoassay

Progesterone is one of the steroid hormones. The hormone is especially important in preparing the uterus for the implantation of the blastocyst and in maintaining pregnancy. Its concentration in serum is measured to determine ovarian function and to predict early pregnancy. The progesterone concentration is also important for in-vitro fertilization and embryo-transfer outcomes. We have established isotope dilution liquid chromatography-tandem mass spectrometry as a primary method for the measurement of progesterone in human serum. Progesterone and its isotopic analogue, progesterone-(13)C(2), in serum were monitored at mass transitions of m/z 315.2/109.2 and 317.2/111.2 respectively in multiple-reaction monitoring (MRM) mode with electrospray positive ionization. For validation of the method, progesterone in a National Institute of Standards and Technology standard reference material (NIST SRM) was measured, and the measured results were in good agreement with the reference values within the uncertainty. On the basis of the established method, progesterone certified reference material (CRM) was developed in this work. The certified value was (1.41 ± 0.036) μg kg(-1). The repeatability of 1.1% and reproducibility of 0.14% showed that ID LC-MS-MS is a reliable and reproducible method. The expanded uncertainty for the measurement of progesterone in the CRM was approximately 2.6% within 95% confidence limits. The detection limit of progesterone was approximately 0.6 μg kg(-1). The progesterone CRMs were distributed to representative clinical laboratories in the Republic of Korea for comparison with the chemiluminescence immunoassay (CLIA), which is the most sensitive immunoassay method. The results from the comparison showed quite a large bias among the participating laboratories. This implies that the CRM is a very important material for establishment of traceability to its practical use.

Final report on CCQM-K63.a,b: Non-peptide hormones in serum: cortisol and progesterone

Hormones are chemical messengers that regulate many life functions. Deviations from normal hormone levels can have serious health consequences. Accurate measurement of hormone levels in serum can be beneficial in diagnosing, monitoring, and treating a number of diseases. Two steroid hormones, cortisol and progesterone, were selected by the Organic Analysis Working Group (OAWG) to evaluate its member Institutes' measurement capabilities for this important class of measurand.Serum concentrations of cortisol range from 30 ng/mL to 230 ng/mL. Serum concentrations of progesterone in adult females range from 0.15 ng/mL to 25 ng/mL but can rise to ≈230 ng/mL during pregnancy. The ability to measure cortisol is indicative of a laboratory's ability to measure steroid hormones at concentration levels similar to cortisol. The ability to measure progesterone is indicative of a laboratory's ability to measure steroid hormones with similar functional groups and concentration levels, such as testosterone.Pilot studies CCQM-P77.a and CCQM-P77.b on the determination of cortisol and progesterone in human serum were completed in 2006. There was good agreement among the results reported by participants who used isotope dilution/mass spectrometry (ID/MS) with either gas chromatography (GC) or liquid chromatography (LC). In 2007 the OAWG decided to proceed with key comparison (KC) CCQM-K63.a, cortisol in human serum, and CCQM-K63.b, progesterone in human serum. Thus, following established OAWG procedure, only results from participants that (1) used an ID/MS-based method, (2) participated in the relevant pilot study, and (3) used a metrologically traceable primary standard were to be eligible for use in calculating the key comparison reference value (KCRV) for each measurand.Six laboratories participated in CCQM-K63.a and eight laboratories participated in CCQM-K63.b. The same pooled frozen female serum material was used in both of the KCs. The mean value for the six ID/MS-based cortisol results is 100.4 ng/g with a relative standard deviation (%RSD) of 1.1%. The mean value for the seven ID/MS-based progesterone originally reported results is 2.83 with a %RSD of 1.8%. While a number of the reported results were not eligible to be used in establishing the KCRVs, the KCRVs as estimated from just the eligible results agree very well with these means. The excellent among-participant agreement indicates that ID/MS-based measurement procedures can provide precise and true results for steroid hormones at levels greater than about 1 ng/g. The progesterone result reported by a laboratory using a non-isotopically labelled internal standard was about 11% larger than the KCRV.Main text.To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/.The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum, bovine serum and bovine milk

Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5–1.0%), adequate sensitivity (0.13–0.16 ng/ml) and recovery (85–115%) for all the three sample matrices, human serum, bovine serum and bovine milk.

Development of ELISA for Measurement of Progesterone Employing 17-α-OH-P-HRP as Enzyme Label

The present study was aimed to develop a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) to measure progesterone in human serum using a heterologous combination of immunogen and enzyme conjugate. The antiserum was raised against Progesterone-3-O-carboxymethyloxime bovine serum albumin (P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-α-hydroxy-progesterone-3-O-carboxymethyloxime (17-α-OH-P-3-O-CMO) with Horseradish Peroxidase (HRP) to form 17-α-OH-P-3-CMO-HRP. A Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 100µL enzyme conjugate along with 50µL of standards in the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100µL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The lowest detection limit of the assay was 0.2 ng/mL. Cross-reaction with analogous steroids pregnenolone and 17-α-OH-P were found to be 6.8 and 6.1%, respectively. For other analogous steroids, it was less than 0.1%. The intra- and inter-assay coefficient of variation ranges from 4.52–7.39% and 4.65–9.55%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.91 (n = 40).

Development of vial wall sorptive extraction and its application to determination of progesterone in human serum

A novel sample preparation method, vial wall sorptive extraction (VWSE), which uses a vial whose internal wall is coated with polydimethylsiloxane (PDMS), was developed. The method was applied to the determination of progesterone in human serum sample. Human serum sample (0.5 mL) spiked with progesterone- 13C 2 was pipetted into the VWSE device and vortex mixing was performed for 30 min. Then, the serum sample was removed and the vial rinsed with purified water. Fifty microliter of methanol as liquid desorption (LD) solvent was pipetted into the VWSE device and vortex mixing was performed for 10 min. Then, the extract was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The correlation coefficient ( r) of the calibration curve over the concentration range of 0.5–200 ng mL −1 was 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 and 0.5 ng mL −1, respectively. The relative recoveries were 97.9% (RSD: 4.4%, n = 6) and 102.8% (RSD: 1.1%, n = 6) for progesterone spiked at 5 and 50 ng mL −1, respectively. This simple, accurate, sensitive, and selective analytical method is applicable to the trace analysis of a minute amount of sample.

Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography–mass spectrometry for the measurement of progesterone in human serum

Miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography–mass spectrometry (GC–MS) for the determination of progesterone in human serum sample is described. The detection limit and the quantification limit of progesterone in human serum sample are 0.5 and 2 ng mL −1 (ppb), respectively. The calibration curve for progesterone is linear with a correlation coefficient of >0.999 in the range of 2–500 ng mL −1. The average recoveries of progesterone in human serum samples spiked with 5, 50 and 200 ng mL −1 progesterone are 97.4% (R.S.D.: 9.3%), 100.3% (R.S.D.: 4.7%) and 99.8% (R.S.D.: 3.4%), respectively. This simple, accurate, sensitive and selective analytical method can be applicable for the determination of progesterone in human serum samples.

Micro-plate Chemiluminescence Enzyme Immunoassay for Clinical Determination of Progesterone in Human Serum

A high-throughput, simple and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the clinical determination of progesterone in human serum, using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP). The solid phase of anti-progesterone antibody was prepared by immunoreaction between anti-progesterone polyclonal antibody and donkey anti-rabbit IgG, i.e. second antibody, which had been physically absorbed on the wells of polystyrene microplate and was used as a universal solid phase. The effect of various factors, such as the dilution of immunoreagents, chemiluminescence substrate, chemiluminescence reaction time and incubation condition were examined and optimized. The optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate were 1:10000 and 1:15000, respectively. The II substrate was chosen and the luminescence was determined after 10 min incubation. The immunoreacted sample was incubated in water bath at 37°C for 1 h. The assay was evaluated with sensitivity as low as 0.08 μg l −1. The relative standard deviation (RSD) was less than 15% for both intra- and inter-assay precision. The recoveries of three different spiked concentration samples were 101%, 101% and 94.4%, respectively. This proposed method has been successfully applied to the clinical evaluation of progesterone in 36 human sera. The results showed a good correlation with the accredited radioimmunoassay (RIA) with a correlative coefficient of 0.9502.

Development of a high‐throughput, indirect antibody immobilization format chemiluminescence enzyme immunoassay (CLEIA) for the determination of progesterone in human serum

A high-throughput and simple chemiluminescence (CL) enzyme immunoassay (CLEIA) for the determination of progesterone (P) in human serum was developed, with the highly sensitive 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD)-alkaline phosphatase (ALP) system as the CL detection system. The results showed that the indirect immobilization of rabbit anti-progesterone polyclonal antibody (RAPA) through secondary antibody exhibited apparent advantages over direct coating in terms of antibody saving and improvement of the coating stability and uniformity. The direct analysis of P in human serum without extraction was realized by using 8-anilino-1-naphthalenesulphonic acid (ANS) to displace P from its binding proteins. The effect of several relevant parameters of the immunoreaction were examined and optimized. Compared with some commercial progesterone kits, the presented CLEIA has higher sensitivity with detection limitation as low as 0.06 ng/mL. The recoveries were 95.9-101%. The coefficient of variation was <8.4% and 9.9% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of P in human serum.

Development and Evaluation of a Candidate Reference Measurement Procedure for the Determination of Progesterone in Human Serum Using Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry

Progesterone is a steroid hormone that is involved in regulating female reproductive processes. Its concentration in blood is measured to determine ovarian function. A candidate reference measurement procedure for progesterone in human serum involving isotope dilution coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The progesterone along with its internal standard (progesterone-13C2) was extracted from the serum matrix using liquid-liquid extraction prior to reversed-phase LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this method on a lyophilized human serum reference material for progesterone [Certified Reference Material (CRM) 347] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added progesterone. The results of this method for progesterone agreed with the certified value within the uncertainty of the measurements for the CRM 347. The recovery of the added progesterone ranged from 100.1 to 100.9%. This method was applied to the determination of progesterone in frozen serum samples from three individual female donors with the progesterone concentrations ranging from 0.151 to 24.42 ng/g. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.1 to 1.4%, and between-set CVs ranging from 0.3 to 0.5%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9998 to 1.0000. The detection limit at a signal-to-noise ratio of approximately 3 was 1.8 pg of progesterone. This well-characterized LC/MS/MS method for serum progesterone, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for progesterone can be compared and that will serve as a standard of higher order for measurement traceability.

Studies on the Influence of the Structural Modifications in the Tracer on the Immunoassay of Progesterone

The main objective of the present study was to examine the influence of different bridges in radioiodinated tracers on the assay performance of progesterone using antibodies. Three homologous and two heterologous immunoassay systems for the measurement of progesterone in human serum are described. Using an antiserum raised against progesterone‐11α‐hemisuccinate–bovine serum albumin (BSA), assays with homologous radioligands, namely progesterone‐11α‐hemisuccinate‐125I‐tyrosine methyl ester (TME) and progesterone‐11α‐hemisuccinate‐125I‐histamine, heterologous bridge radioligand, namely progesterone‐11α‐hemiphthalate‐125I‐TME, and a heterologous site radioligand namely progesterone‐3‐(O‐carboxymethyl) oxime (CMO)‐125I‐histamine were optimized. A homologous assay system, using antiserum raised against progesterone‐3‐carboxymethyl oxime‐BSA and progesterone‐3‐CMO‐125I‐histamine as the radioligand was also optimized to develop a radioimmunoassay (RIA) for serum progesterone. Amongst the two homologous radioligands, viz., progesterone‐11α‐hemisuccinate‐125I‐histamine and the corresponding TME conjugate tracer, the former yielded a standard curve with a higher slope (−0.6) as compared to the latter (−0.5). The heterologous bridge system with progesterone‐11α‐hemiphthalate‐125I‐TME resulted in a more sensitive assay (slope of −0.8) than the homologous tracers, whilst the heterologous site radioligand, viz., progesterone‐3‐CMO‐125I‐histamine gave the most sensitive assay (slope of −1.2). The homologous assay with antiserum against progesterone‐3‐CMO‐BSA and progesterone‐3‐CMO‐125I‐histamine tracer gave a standard curve having a slope of –0.97. The two antibodies developed against progesterone, viz., progesterone‐11α‐hemisuccinate‐BSA and progesterone‐3‐CMO‐BSA were characterized for their titre, sensitivity, and specificity. Considering the slope, sensitivity, cross‐reactivity, and the quality of tracer, the assay system using antiserum against progesterone‐11α‐hemisuccinate‐BSA and progesterone‐3‐CMO‐125I‐histamine was found to be suitable for the development of RIA for serum progesterone. The bridges used in an immunogen for production of antibodies, as well as in the preparation of tracer, have a great influence on the assay characteristics.

Simultaneous quantitative determination of norgestrel and progesterone in human serum by high-performance liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A selective, reliable and rapid method for the simultaneous determination of progesterone and norgestrel concentrations in human serum after taking oral contraceptive tablet has been developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) with atmospheric pressure chemical ionization (APCI) interface. The norethisterone was used as the internal standard. Selected transitions of m/z 313/245 for norgestrel, m/z 315/97 for progesterone and m/z 299/109 for norethisterone were monitored using multiple reaction monitoring (MRM) mode for quantitation. The assay was linear over the concentration range of 0.2-50 ng mL-1 for norgestrel and progesterone. The lower level of quantitation in human serum was obtained at 0.2 ng mL-1 for both norgestrel and progesterone using optimum tuning parameters. The intra-assay precision and inter-assay precision do not exceed 10 and 9%, respectively. The method has been applied to the determination of norgestrel and progesterone in serum of female volunteers.

Development of a direct radioimmunoassay for serum progesterone

A direct radioimmunoassay for the measurement of progesterone in human serum is described. Progesterone 11α-hemisuccinate was conjugated to tyrosine methyl ester (TME) by the mix anhydride method and then iodinated using chloramine-T. The radiochemical purity of different batches of125I-progesterone was greater than 95% and showed 70–75% binding with excess antibody. Progesterone 11α-hemisuccinate was coupled with BSA and injected to rabits. Antisera collected after three booster injections and having aK value of 1·1091/M was selected for the assay. Significant reduction in binding with antibody was seen when hormone free serum was used in the assay system. Various blocking agents were tried to reduce the serum effects and none of them were found satisfactory. From a series of optimization experiments, an assay was developed without the use of blocking agents. This assay used a much higher concentration of antibody along with lower amount of serum sample (50 μl). The optimized assay has a sensitivity of 0.5 nj/ml and a working range of 0.5 to 100 ng/ml. Serum samples were analyzed analysis showed good correlation between the results obtained from the present system and the DPC kit. (Y=0.93X+0.5,r=0.93, forn=25).

Release of protein bound progesterone in human serum by ioglycamide.

With the use of an aqueous two-phase system the influence of ioglycamide on the serum protein binding of progesterone was investigated. Ioglycamide was found to cause a decrease in the association constants for the binding of progesterone to both albumin and transcortin. The binding capacity for the progesterone binding to transcortin was also decreased. The decreased progesterone binding was discussed in terms of clinical relevance in connection with adverse reactions at cholegraphy with intravenous injection of ioglycamide.