Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11ß-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability.

Background Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11ß-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability. Methods An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis. Results The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively. Conclusions We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.

Oestradiol measurement during fulvestrant treatment for breast cancer.

Biochemical evaluation of menopausal status is used to inform treatment decisions, including clinical trial eligibility in women with oestrogen receptor positive breast cancer. However, fulvestrant may interfere with oestradiol immunoassays and confound accurate assessment in this context. We conducted a service evaluation of two immunoassays and an LC-MS/MS assay to determine the extent of the interference. Serum oestradiol levels were analysed by two immunoassays (Siemens Centaur XP and Abbott Architect) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). Immunoassay gave higher serum oestradiol results than LC-MS/MS at low concentrations, with improved analytical sensitivity demonstrated by LC-MS/MS. Cross-reactivity of fulvestrant was observed for each immunoassay. We have shown that two commonly used immunoassays do not demonstrate the required sensitivity or specificity for the measurement of oestradiol in a breast cancer population. For patients receiving fulvestrant, spurious results may be generated that could impact treatment decisions. LC-MS/MS is recommended in this setting.

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS.

BACKGROUND: Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. METHOD: Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. RESULTS: Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%. CONCLUSIONS: The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.

Serum Cortisol: An Up-To-Date Assessment of Routine Assay Performance.

BACKGROUND: Accurate serum cortisol quantification is required for the correct diagnosis and management of adrenal pathologies. Presently, most laboratories use immunoassay to measure serum cortisol with proficiency schemes demonstrating a wide dispersion of results. Here, we investigate the effects of sex, matrix, and antibody specificity on serum cortisol quantification in 6 routine assays. METHODS: Surplus serum was obtained before disposal and the following cohorts were created: males, nonpregnant females, pregnant patients, and patients prescribed either metyrapone or prednisolone. Samples were anonymized and distributed to collaborating laboratories for cortisol analysis by 6 routine assays. Cortisol was also measured in all samples using an LC-MS/MS candidate reference measurement procedure (cRMP); cortisol-binding globulin (CBG) was measured in the nonpregnant and pregnant female cohorts. RESULTS: Considerable inter- and intraassay variation was observed across the male and nonpregnant female cohorts relative to the cRMP. Four immunoassays underrecovered cortisol in the pregnancy cohort, and CBG was found to be significantly higher in this cohort than in the nonpregnant females. In the metyrapone and prednisolone cohorts, all immunoassays overestimated cortisol. The first generation Roche E170 and Siemens Centaur XP were particularly prone to overestimation. In all cohorts the routine LC-MS/MS assay aligned extremely well with the cRMP. CONCLUSIONS: Despite the clinical importance of serum cortisol, the performance of routine immunoassays remains highly variable. Accurate quantification is compromised by both matrix effects and antibody specificity. Underpinning this study with a cRMP has highlighted the deficiencies in standardization across routine cortisol immunoassays.

Evaluation of an Axial Pattern Flap Based on the Cranial Cutaneous Branch of the Saphenous Artery: A Cadaveric Perfusion Study.

OBJECTIVES: To confirm the anatomic location of the cranial cutaneous branch of the saphenous artery (CCSA), delineate the angiosome of the CCSA, and provide guidelines for clinical use of an axial pattern flap based on the CCSA. STUDY DESIGN: Anatomic study. ANIMALS: Greyhound cadavers (n=10). METHODS: Shortly after euthanasia, the CCSA was identified and isolated in each hindlimb. Methylene blue and radiographic perfusion studies were performed. The skin was freed from the thigh for photographic and radiographic images. The dimensions of the skin area suitable for use as an axial pattern flap were related to anatomical landmarks. Mock surgical elevation and transposition of the flap in 2 dogs allowed assessment of flap mobility and ease of donor site closure for clinical use. RESULTS: The CCSA was reliably identified in all dogs branching from the saphenous artery as it became superficial to the sartorius muscle, immediately distal to the caudal cutaneous branch and proximal to the genicular branches. Genicular branches were variably paired or singular. Perfusion studies defined the CCSA angiosome as the area cranial to the saphenous artery and caudal to the cranial border of the thigh, extending proximally from the level of the medial tibial condyle to two-thirds of the distance to the inguinal ring. CONCLUSION: An axial pattern flap based on the CCSA could be expected to cover skin defects of the cranial aspect of the distal thigh and stifle, the popliteal region caudal to the stifle, and the proximal medial crus.

Persistent urinary incontinence in female Golden Retrievers following laser ablation of intramural ectopic ureters may be associated with the presence of historical urinary tract infection.

OBJECTIVE: To identify predictive factors for postoperative continence in female Golden Retrievers following cystoscopic-guided laser ablation of intramural ectopic ureters (CLA-EU). ANIMALS: 41 client-owned female entire Golden Retrievers with uni- or bilateral intramural ectopic ureter(s) were retrospectively enrolled. METHODS: Patients were diagnosed with ectopic ureters with a combination of ultrasonography and cystoscopy. CLA-EU was performed for all dogs so that each ureteral opening was considered to be in an appropriate position by a single operator. All dogs had short-term follow-up 4 weeks and long-term follow up > 10 weeks after the procedure via telephone, which included urinary continence scoring. Clinical factors and ultrasonographic and cystoscopic findings from initial presentation were evaluated to identify predictive factors for postoperative continence. RESULTS: Short-term urinary continence was achieved in 46.3% of dogs with no additional medical therapies. Presence of historical urinary tract infections prior to CLA-EU (OR, 0.130; 95% CI, 0.020 to 0.621; P = .018) was negatively correlated and ureteral dilatation (OR, 34.260; 95% CI, 1.813 to 2,143; P = .043) was positively correlated with likelihood of urinary continence. Long-term urinary continence was achieved in 63.4% of dogs, and presence of historical urinary tract infections was negatively prognostic (OR, 0.173; 95% CI, 0.023 to 0.856; P = .048). CLINICAL RELEVANCE: Female Golden Retrievers undergoing CLA-EU have similar outcomes to those reported for other mixed-breed cohorts with > 30% of dogs failing to regain urinary continence. Historical urinary tract infections were significantly associated with both short- and long-term urinary continence in our population.

Serum cortisol assay performance following the 1 mg overnight dexamethasone suppression test.

BACKGROUND: The 1 mg overnight dexamethasone suppression test (ONDST) is recommended for the differential diagnosis of Cushing's syndrome and the investigation of adrenal incidentalomas. Despite documented variation in serum cortisol immunoassay performance, little has been published regarding its effect on the ONDST. AIMS: Assess the performance of three immunoassay platforms (Roche Elecsys II, Abbott Alinity & Siemens Centaur) when compared to a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. METHODS: Samples (n = 77) sent to the laboratory as part of an ONDST were retrieved prior to disposal, anonymized, and analysed on all platforms. Samples with factors impacting immunoassay analysis quality were excluded. Results were statistically compared to an LC-MS/MS method that previously demonstrated excellent comparability to a candidate reference method. RESULTS: The Roche gen II showed a mean bias of -2.4 nmol/L and a Passing-Bablok fit of y = -0.9 + 0.97x. This was not affected by sex. The Abbott showed a mean bias -18.8 nmol/L, and a fit of y = -11.3 + 0.88x. This bias was -20.7 nmol/L in females versus -17.2 nmol/L in males. The Siemens had a mean bias of 2.3 nmol/L and a fit of y = 1.4 + 1.07x. This bias was 5.7 nmol/L in males versus -1.0 nmol/L in females. CONCLUSIONS: Clinicians should be aware of the method-dependent variation that exists within serum cortisol analysis during the ONDSTs. Roche and Siemens aligned more closely with LC-MS/MS while the Abbot may cause a reduction in ONDST sensitivity. This data supports assay-specific cut-offs for the ONDST.

Designing calcium release channel inhibitors with enhanced electron donor properties: stabilizing the closed state of ryanodine receptor type 1.

New drugs with enhanced electron donor properties that target the ryanodine receptor from skeletal muscle sarcoplasmic reticulum (RyR1) are shown to be potent inhibitors of single-channel activity. In this article, we synthesize derivatives of the channel activator 4-chloro-3-methyl phenol (4-CmC) and the 1,4-benzothiazepine channel inhibitor 4-[-3{1-(4-benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (K201, JTV519) with enhanced electron donor properties. Instead of activating channel activity (~100 µM), the 4-methoxy analog of 4-CmC [4-methoxy-3-methyl phenol (4-MmC)] inhibits channel activity at submicromolar concentrations (IC(50) = 0.34 ± 0.08 µM). Increasing the electron donor characteristics of K201 by synthesizing its dioxole congener results in an approximately 16 times more potent RyR1 inhibitor (IC(50) = 0.24 ± 0.05 µM) compared with K201 (IC(50) = 3.98 ± 0.79 µM). Inhibition is not caused by an increased closed time of the channel but seems to be caused by an open state block of RyR1. These alterations to chemical structure do not influence the ability of these drugs to affect Ca(2+)-dependent ATPase activity of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase type 1. Moreover, the FKBP12 protein, which stabilizes RyR1 in a closed configuration, is shown to be a strong electron donor. It seems as if FKBP12, K201, its dioxole derivative, and 4-MmC inhibit RyR1 channel activity by virtue of their electron donor characteristics. These results embody strong evidence that designing new drugs to target RyR1 with enhanced electron donor characteristics results in more potent channel inhibitors. This is a novel approach to the design of new, more potent drugs with the aim of functionally modifying RyR1 single-channel activity.

Measurement of salivary cortisol with liquid chromatography-tandem mass spectrometry in patients undergoing dynamic endocrine testing.

OBJECTIVE: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) eliminates cross-reactivity, which is a major limitation of immunoassays used for the measurement of salivary cortisol (SalC). We aimed to evaluate the potential of SalC measured by LC-MS/MS in patients undergoing assessment of the HPA axis. DESIGN AND PATIENTS: Cross-sectional study of 78 patients admitted for routine testing in a specialized endocrine unit. MEASUREMENTS: Matched serum and saliva samples were collected from 68 patients who had a short synacthen test (SST, 250 mcg im) and 10 patients who had an insulin tolerance test (ITT, insulin 0.15 U/kg iv). Serum cortisol (SerC) was measured with an automated immunoassay and SalC with LC-MS/MS. Adequate SerC responses were >500 nmol/l. RESULTS: In all patients with adequate responses, the relative increase in SalC was significantly higher than that in SerC [6.4(0.3-26.1) vs. 1.0(0.3-4.9), P < 0.0001)]. The SerC-SalC relationship was better explained by an exponential rather than a linear model (R(2)=0.83 vs. R(2)=0.65, both P < 0.0001). Based on 59 patients with adequate SerC responses to an SST, an adequate SalC response was defined as 8.3 nmol/l. Seven patients following an SST and three patients following an ITT showed inadequate responses in both SerC and SalC, but two patients with CBG deficiency showed a low SerC with normal SalC. CONCLUSIONS: We have shown an excellent diagnostic sensitivity and specificity of LC-MS/MS SalC in the assessment of the HPA axis and superiority over SerC when CBG levels are altered. The exponential relationship between SerC and SalC supports the concept of CBG binding capacity saturation.

Efficacy of adhesive incise drapes in preventing bacterial contamination of clean canine surgical wounds.

OBJECTIVE: To investigate the efficacy of adhesive incise drapes in reducing bacterial contamination of clean canine surgical wounds. STUDY DESIGN: Randomized clinical trial. ANIMALS: Dogs (n=100) having elective ovariohysterectomy or stifle surgery. METHODS: Dogs were randomly assigned to 1 of 2 groups: drape or no drape. Swabs obtained from the inner edge of the surgical wound at the beginning (swab 1) and end (swab 2) of surgery were submitted for microbial culture. Number of colony forming units was counted for all positive cultures and change in bacterial counts between swabs 1 and 2 was calculated. Percentage adhesive drape adherence at the end of surgery was calculated from a digital photograph of the surgical site. duration of surgery/anesthesia and the anesthetic induction agent used were recorded. RESULTS: There was a significant increase in bacterial counts between swabs 1 and 2 (P=.001). Wound contamination was 14% (6 drape; 8 no drape; P=0.78) with Staphylococcus spp. most commonly isolated. Median percentage drape adherence at the end of surgery was 89.3% (0-100%). Duration of anesthesia was significantly related to wound contamination (P=.013), but duration of surgery and anesthesia induction agent were not. CONCLUSIONS: Adhesive incise drapes did not reduce wound contamination of clean canine surgical wounds. CLINICAL RELEVANCE: Use of adhesive incise drapes in clean surgical procedures is of questionable benefit in dogs.

State-of-the-art of serum testosterone measurement by isotope dilution-liquid chromatography-tandem mass spectrometry.

BACKGROUND: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine. METHODS: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs. RESULTS: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%. CONCLUSIONS: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

Development of a rapid liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone and its clinical verification.

Background Measurement of serum dexamethasone during the overnight dexamethasone-suppression test has been recommended to reduce false-positive results when investigating Cushing's syndrome or increasingly commonly found adrenal incidentalomas. Despite this, there remains a paucity of well-validated dexamethasone methods currently available. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry serum dexamethasone assay and verify its utility in a cohort of postmenopausal females. Method Isotopically labelled internal standard was added to samples prior to supported liquid extraction. Extracts were analysed using liquid chromatography tandem mass spectrometry in the positive electrospray ionization mode. Multiple reaction monitoring was used to detect dexamethasone and its corresponding internal standard transitions. Normal healthy postmenopausal women ( n = 95) were recruited and underwent an overnight dexamethasone suppression test, with serum dexamethasone and cortisol measurements at 09:00 after administration of oral dexamethasone 1 mg at 23:00 the night before. Results Mean intra- and inter-assay imprecision were 4.1% and 2.9%, respectively, for dexamethasone concentrations of 1.5, 6.0 and 12.0 nmol/L. Matrix effects were found to be negligible at 106-109% with recovery ranging from 96 to 100%. The limit of quantitation was 0.25 nmol/L, and structural analogue analysis proved the method to be robust against interferences. Applying a serum dexamethasone cut-off of >3.3 nmol/L was associated with a serum cortisol ≤50 nmol/L in 84/95 individuals. Conclusion We have developed a sensitive and robust liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone. The method can be used to identify false-positive results during the overnight dexamethasone suppression test or for pharmacokinetic studies.

A simplified method for the measurement of urinary free cortisol using LC-MS/MS.

The measurement of 24 h urinary free cortisol is used in the investigation of patients with symptoms of hypercortisolism. Many different methods have been published for the measurement of cortisol, but most of these methods involve cumbersome pre-extraction of the cortisol prior to analysis. We have developed a method using in-well protein precipitation which serves to clean up the sample without requiring lengthy sample preparation. A Shimadzu SIL-HT autosampler was used to inject 50 microL of extract onto a Phenomemex Gemini C18 guard column attached to a Waters Xbridge C18 column. The eluant was introduced directly into a Waters Quattro Micro tandem mass spectrometer. The method was found to be linear up to 3448 nmol/L with a lower limit of detection of 5.3 nmol/L. Precision and accuracy were acceptable, and no interference was noted from compounds such as prednisolone or fenofibrate. This assay was compared to a previously published method, which uses solid phase extraction prior to LC-MS/MS analysis. We have developed a simplified, robust assay for the quantitation of urinary free cortisol that will increase the throughput of the assay and avoid the use of neurotoxic solvents such as dichloromethane.

Validation of a liquid chromatography tandem mass spectrometry assay for serum creatinine and comparison with enzymatic and Jaffe methods.

BACKGROUND: Glomerular filtration rate (GFR) can be estimated using creatinine clearance or calculated formulae, methods which rely on creatinine quantification in serum. Creatinine is most frequently measured using the Jaffe method, but this is prone to interference by, for example, bilirubin, protein and ketones. Recent amendments to this assay have been made by manufacturers in an attempt to compensate for the protein interference. METHODS: We developed a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of creatinine in serum, using a strong cation exchange column to give a short run time of 1.5 min and minimal ion suppression. The method had a lower limit of quantification of 5 micromol/L and the intra- and inter-assay imprecision was <5% and <7%, respectively. Serum samples (n = 182) were subsequently analysed by enzymatic, compensated Jaffe and LC-MS/MS methods and results compared. RESULTS: The compensated Jaffe and enzymatic methods gave similar results to the LC-MS/MS method at concentrations below 150 micromol/L. At concentrations above this value, creatinine was underestimated by both the compensated Jaffe and enzymatic methods compared with the LC-MS/MS method. CONCLUSIONS: We have developed a novel, simple, sensitive LC-MS/MS assay for use in comparative studies or when the results of either Jaffe or enzymatic assays are in question.

Lingual haemangiosarcoma in a crossbred dog.

An eight-year-old, male neutered, crossbred dog was presented for investigation of a lingual mass of four months duration. Oral examination revealed a 7 cm × 5 cm soft, fluctuant mass at the caudal aspect of the tongue. Ultrasound examination of the mass demonstrated mixed echogenicity, with cavitations containing hypoechoic and anechoic regions. Lingual haemangiosarcoma was diagnosed on histopathological examination of multiple biopsy samples, with confirmation of the vascular endothelial origin of tumour cells by positive immunolabelling for factor VIII-related antigen.

A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by liquid chromatography tandem mass spectrometry.

BACKGROUND: The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens. METHODS: Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer. RESULTS: Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%. DISCUSSION: We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application.

Liquid chromatography tandem mass spectrometry in the clinical laboratory.

Clinical laboratory medicine has seen the introduction and evolution of liquid chromatography tandem mass spectrometry in routine clinical laboratories over the last 10-15 years. There still exists a wide diversity of assays from very esoteric and highly specialist manual assays to more simplified kit-based assays. The technology is not static as manufacturers are continually making improvements. Mass spectrometry is now commonly used in several areas of diagnostics including therapeutic drug monitoring, toxicology, endocrinology, paediatrics and microbiology. Some of the most high throughput analyses or common analytes include vitamin D, immunosuppressant monitoring, androgen measurement and newborn screening. It also offers flexibility for the measurement of analytes in a variety of different matrices which would prove difficult with immunoassays. Unlike immunoassays or high-pressure liquid chromatography assays using ultraviolet or fluorescence detection, mass spectrometry offers better specificity and reduced interferences if attention is paid to potential isobaric compounds. Furthermore, multiplexing, which enables multiple analytes to be measured with the same volume of serum is advantageous, and the requirement for large sample volumes is decreasing as instrument sensitivity increases. There are many emerging applications in the literature. Using mass spectrometry to identify novel isoforms or modified peptides is possible as is quantification of proteins and peptides, with or without protein digests. Future developments by the manufacturers may also include mechanisms to improve the throughput of samples and strategies to decrease the level of skill required by the operators.

A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry.

BACKGROUND: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum oestradiol and oestrone. The assay uses a relatively small volume and has a rapid run time. METHODS: Supported liquid extraction was performed on 250 µL of sample using methyl tertiary butyl ether. The extract was dried and reconstituted with 100 µL of 40% methanol. Online automated solid phase extraction was performed on 75 µL of extract using C18 cartridges on a Waters OSM coupled to a Waters TQS mass spectrometer. Serum samples (n = 197) were analysed by LC-MS/MS and a commercial immunoassay. RESULTS: The lower limit of quantitation for oestradiol and oestrone was 10 and 6 pmol/L, respectively. The coefficient of variation (CV) of the assay for oestradiol and oestrone concentrations of 125 pmol/L was <7%. The assay had a CV of 10% at 22 pmol/L for oestradiol and 5% at 16 pmol/L for oestrone. The average recovery for oestradiol was 102% and oestrone was 106%. The comparison with a commercial immunoassay gave the following equation: Immunoassay = 0.94 × LC-MS/MS + 21 pmol/L. The run time was 4.5 min per sample. DISCUSSION: We have developed a rapid assay for the LC-MS/MS measurement of oestradiol and oestrone which does not require derivatization in the sample preparation. The assay is suitable for routine clinical use or for clinical trials. The assay demonstrated superior performance compared to immunoassays at lower concentrations making it more suitable for use in males and patients on aromatase inhibitors.