Sweat Cortisol and Cortisone Determination in Healthy Adults: UHPLC-MS/MS Assay Validation and Clinical Application.

A simple and effective ultra-high-performance liquid chromatography assay linked to tandem mass spectrometry (UHPLC-MS/MS) for measuring cortisol and cortisone levels in human sweat has been developed and validated. A noninvasive world standard sweat collecting equipment was utilized to collect samples. The samples were analyzed using an Atlantis dC18 (2.1 × 100 mm, 3 µm) column with a 2 mM ammonium acetate and acetonitrile (1 : 1, v : v) mobile phase. In an isocratic condition, the mobile phase was delivered at a flow rate of 0.3 ml/minute. A positive electrospray ionization interface with multiple-reaction monitoring mode was used to provide simultaneous quantification of cortisol, cortisone, and internal standard at transitions of 363.11 to 121.00, 361.18 to 163.11, and 367.19 to 121.24, respectively. The method was validated for cortisol and cortisone determination over a concentration range of 0.5-50 ng/mL The detection limits for cortisol and cortisone in human sweat were 0.3 and 0.2 ng/ml, respectively. The interday coefficients of variation of cortisol and cortisone were ≤8.5% and ≤10.01%, whereas bias was in the range from -7.9% to 2.1% and from -4.3% to 3.0%, respectively. The assay was successfully applied to evaluate the cortisol-to-cortisone ratio in sweat samples collected from healthy adult volunteers.

Quality assessment of nine paracetamol 500 mg tablet brands marketed in Saudi Arabia.

OBJECTIVE: To evaluate in-vitro quality of paracetamol 500 mg tablet brands marketed in Saudi Arabia. RESULTS: Two reference (R1 and R2) and seven generic (G1-G7) brands were commercially available. Four brands were single-drug, containing paracetamol only (R1, G1-G3) and five contained additional active ingredients (R2, G4-G7). All brands were immediate-release. Weight variation (n = 20, range as percent difference from mean), active substance content (n = 20, mean (SD) as percent difference from label), breaking force (n = 10, mean (SD)), and friability (n = 20, as percent weight loss) ranged from 97 to 102%, 96.1% (2.9%) to 99.8% (1.1%), 9.9 (0.4) to 21.0 (0.9) kg, and 0.017% to 0.809%, respectively. Disintegration (water medium) time (n = 6, minute: second) ranged from 02:35-03:09 to 12:49-13:10. Dissolution (phosphate buffer, pH 5.8) profile showed a mean release at 30 min of 87% to 97% of label content, with seven brands passing stage-1 (≥ 85% for each of 6 test units) and two passing stage-2 (mean of 12 test units ≥ 85%) criteria. Despite statistically significant differences between R1 and R2 and some of their corresponding generic brands in active substance content, breaking force, and amount dissolved at 30 min, all nine brands met the pre-specified quality standards.

Misleading localization by 18F-fluorocholine PET/CT in familial hypocalciuric hypercalcemia type-3: a case report.

BACKGROUND: Familial hypocalciuric hypercalcemia (FHH) is a heterogeneous autosomal-dominant disorder of calcium hemostasis that may be difficult to distinguish clinically from mild primary hyperparathyroidism. Loss-of-function mutations mainly involving Arg15 residue of the adaptor-related protein complex 2, sigma subunit 1 (AP2S1) cause a rarer, more recently recognized form of FHH, FFH type-3. Recently, 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) showed superior sensitivity to conventional imaging in localizing parathyroid adenomas. We report a new FFH type-3 patient who underwent unnecessary parathyroidectomy in association with misleading FCH-PET/CT imaging. CASE PRESENTATION: A 29-year old woman was initially evaluated for parathyroid hormone (PTH)-dependent hypercalcemia in 2013. Medical history was positive only for chronic constipation and malaise with no personal or family history of hypercalcemia, kidney stones, or neck surgery. Over seven years, serum calcium level was 2.51-2.89 mmol/L with concomitant PTH level of 58.7-94.8 mmol/L. Serum phosphate levels were in the low/low normal range. Serum creatinine and magnesium levels were normal. 25-hydroxy vitamin D level was 13 nmol/L. 24-hour urine calcium level was 1.92 mmol/day but increased to 6.99 mmol/day after treatment with cholecalciferol 1000 IU daily. Bone mineral density and renal ultrasound were normal. Parathyroid ultrasound showed two hypoechoic nodules inferior to the left and right thyroid lobes; however, 99mtechnitium-sestamibi scans (2013, 2016, 2018) were negative. FCH-PET/CT (2019) showed focal uptake co-localizing with the nodule inferior to the left thyroid lobe. The patient underwent left inferior parathyroidectomy and pathology was consistent with parathyroid hyperplasia. However, postoperatively, serum calcium and PTH levels remained elevated and FCH-PET/CT and ultrasound showed persistence of the uptake/nodule. Whole exome sequencing showed Arg15Cys mutation in the AP2S1 gene characteristic of FHH type-3. CONCLUSIONS: In this new case of FHH type-3, FCH-PET/CT failed to localize to the hyperplastic parathyroid glands and localized instead to apparently a lymph node. This, together with increased urinary calcium after vitamin D supplementation, led to unnecessary parathyroidectomy. Given the increasingly lower cost of genetic testing and the cost of follow up and unnecessary surgery, it may prudent to include genetic testing for FHH early on in patients with mild PTH-dependent hypercalcemia.

Importance Hierarchy of Surrogate Medical Decision Making Determinants: A Q-Methodology Study in Middle Eastern and East Asian Men.

BACKGROUND: Factors other than patient's preference may influence surrogate medical decision making in a culture- and viewpoint-dependent way. We explored the importance hierarchy of potential surrogate medical decision making determinants to Middle-Eastern (ME) and East-Asian (EA) men according to their norm-perception (N-viewpoint), preference as patients (P-viewpoint), and preference as surrogate decision-makers (S-viewpoint). METHODS: Each respondent (120 ME, 120 EA) sorted 28 items reflecting potential determinants into a fixed distribution of importance hierarchy according to the three viewpoints. Latent decision making models were explored by by-person factor analysis (Q-methodology). RESULTS: Six models were identified for each ME and EA viewpoint (total 36). Patient's health-related, patient's preference-related, and society's interests-related determinants were strongly embraced in 34, 3, and zero models and strongly discounted in 2, 5, and 21 models, respectively. Patient's religious/spiritual belief was strongly embraced in 6 EA models compared to 2 ME models and strongly discounted in 2 EA models compared to 5 ME models. Further, family-centric and surrogate's interest-related determinants were strongly embraced in 8 EA models compared to 1 ME model. They were also strongly embraced in 5 P-viewpoint compared to 2 S-viewpoint models and strongly discounted in 4 P-viewpoint compared to 11 S-viewpoint models. Despite the overall predominance of patient's health-related determinants and culture- and viewpoint-dependent differences, Q-methodology analysis identified relatively patient's preference-influenced, religious/spiritual beliefs-influenced, emotion-influenced, and familism-influenced models and showed notable overlap in models. CONCLUSIONS: Patient's health was more important than other potential medical surrogate decision making determinants, including patient's preference, for both ME and EA men and in all viewpoints. The relative importance of some determinants was culture- and viewpoint- dependent and allowed description of different albeit overlapping models.

Pharmaceutical quality of seven brands of diclofenac tablet on the Saudi market.

OBJECTIVE: We previously reported the pharmaceutical quality of eight brands of 50 mg enteric-coated diclofenac sodium tablet available on the Saudi market. Here, we assess the quality of reference (R1) and four generic (G1-G4) brands of 50 mg immediate-release diclofenac potassium tablet and of reference (R2) and generic (G5) brands of 100 mg sustained-release diclofenac sodium tablet. RESULTS: Weight variation (range as % difference from mean), active substance content (mean (SD) as % difference from label), breaking force [mean (SD)], and friability (as % weight loss) were 95-104% and 99-102%, 100.9% (3.4%) and 105.6 (4.2%), 12.2 (1.3) and 12.9 (1.8) kg, and 0.0014% and 0.0012%, for R1 and R2, respectively. For G1-G5, they were ≤ ± 2%, 98.8% (2.7%) to 109.2% (3.8%), 6.4 (0.6) to 13.3 (1.0) kg, and 0.0007% to 0.0261%, respectively. R1 and G1-G4 disintegrated within 04:50-17:20 min: seconds and released a mean of 89-100% of label active substance content by 60 min in buffer (pH 6.8). R2 and G5 did not disintegrate or dissolve in 0.1 N HCl for 2 h, disintegrated in buffer (pH 6.8) in 01:58-02:15 h: minutes, and fulfilled dissolution criteria (pH 7.5) for both United States Pharmacopoeia test-1 and test-2. Thus all seven brands met pre-specified quality criteria.

Factors underlying surrogate medical decision-making in middle eastern and east Asian women: a Q-methodology study.

BACKGROUND: It is not clear how lay people prioritize the various, sometimes conflicting, interests when they make surrogate medical decisions, especially in non-Western cultures. The extent such decisions are perspective-related is also not well documented. METHODS: We explored the relative importance of 28 surrogate decision-making factors to 120 Middle-Eastern (ME) and 120 East-Asian (EA) women from three perspectives, norm-perception (N), preference as patient (P), and preference as surrogate decision-maker (S). Each respondent force-ranked (one to nine) 28 opinion-items according to each perspective. Items' ranks were analyzed by averaging-analysis and Q-methodology. RESULTS: Respondents' mean (SD) age was 33.2 (7.9) years; all ME were Muslims, 83% of EA were Christians. "Trying everything possible to save patient," "Improving patient health," "Patient pain and suffering," and/or "What is in the best interests of patient" were the three most-important items, whereas "Effect of caring for patient on all patients in society," "Effect of caring for patient on patients with same disease," and/or "Cost to society from caring for patient" were among the three least-important items, in each ME and EA perspectives. P-perspective assigned higher mean ranks to family and surrogate's needs and burdens-related items, and lower mean rank to "Fear of loss" than S-perspective (p<0.001). ME assigned higher mean ranks to "Medical facts" and "Surrogate own wishes for patient" and lower mean rank to "Family needs" in all perspectives (p<0.001). Q-methodology identified models that were relatively patient's preference-, patient's religious/spiritual beliefs-, or emotion-dependent (all perspectives); medical facts-dependent (N- and S-perspectives), financial needs-dependent (P- and S-perspectives), and family needs-dependent (P-perspective). CONCLUSIONS: 1) Patient's health was more important than patient's preference to ME and EA women; society interest was least important. 2) Family and surrogate's needs/ burdens were more important, whereas fear of loss was less important to respondents as patients than as surrogate decision-makers. 3) Family needs were more important to EA than ME respondents, the opposite was true for medical facts and surrogate's wishes for patient. 4) Q-methodology models that relatively emphasized various surrogate decision-making factors overlapped the ME and EA women' three perspectives.

Eight enteric-coated 50 mg diclofenac sodium tablet formulations marketed in Saudi Arabia: in vitro quality evaluation.

OBJECTIVE: To evaluate in vitro quality of enteric-coated 50 mg diclofenac sodium tablet formulations on Saudi market. RESULTS: A reference and seven generic (G1-7) formulations were commercially available in December 2019/January 2020 and were assessed within 25-75% of manufacture-expiration period. Weight variation (range as% difference from mean, n = 20), active substance content (ASC, mean (SD) as% difference from label, n = 20), hardness (mean (SD), n = 10), and friability (% weight loss, n = 20) were 97-103%, 102.0% (3.4%), 15.4 (1.1) kg, and 0.24%, respectively, for the reference. For G2-7, they were ≤ ±5%, 98.6% (4.0%) to 109.9% (1.8%), 11.9 (0.9) to 18.3 (0.8) kg, and ≤ 0.00 to 0.75%, respectively. G1 ASC, hardness, and friability were 111.3% (1.7%), 20.1 (1.7) kg, and 1.10%, respectively. Disintegration time (n = 6) and dissolution profile (n = 8) were also determined. No formulation disintegrated or released ˃ 0.1% of label ASC in 0.1 N HCl for 2 h. The reference disintegrated in 15:00 min:seconds and released a mean (range) of 100% (99-103%) of label ASC by 45 min in phosphate buffer (pH = 6.8). G1-7 disintegrated in 8:53 to 20:37 min:seconds and released 81% (69-90%) (G1) to 109%. Except for borderline performance of G1, all formulations passed in vitro quality tests according to United States Pharmacopoeia.

An improved method for measurement of testosterone in human plasma and saliva by ultra-performance liquid chromatography-tandem mass spectrometry.

The aim of the study was to develop and validate a practical assay of clinically relevant testosterone levels in human plasma and saliva. We performed ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis on Atlantis dC18 steel column using a mobile phase of 2-mM ammonium acetate and acetonitrile (20:80, v: v) that was delivered at 0.3 ml/min. After adding d3-testosterone as an internal standard (IS), we extracted plasma and salivary samples with methyl tert-butyl ether. Mass spectrometry was performed in electrospray positive-ion mode. Targeted ion transitions were examined at m/z 289.18 → 97.04 and 292.24 → 97.04 for testosterone and IS, respectively. We validated the method according to the US Food and Drug Administration guidelines. Elution times for testosterone and IS were both around 1.35 min. Testosterone level was linearly associated (r 2 = 0.9975 and 0.9958) with peak area ratio of testosterone to IS between 0.5-50 ng/ml and 10-400 pg/ml in plasma and saliva, respectively. The coefficient of variation and bias were ≤12.6% and ≤±12.1% in plasma and ≤10.2% and ≤±5.3% in saliva. The extraction recovery of testosterone was ≥92% from plasma and ≥94% from saliva. Testosterone was stable (≥91%) for 24 h at room temperature and for 8 weeks at -20°C in both plasma and salivary samples. We report a simple, validated, UPLC-MS/MS assay that can be used to determine clinically relevant levels of testosterone in human plasma and saliva.

Modeling Lay People's Ethical Attitudes to Organ Donation: A Q-Methodology Study.

BACKGROUND: Organ donation is commonly evaluated by biomedical ethicists based largely on principlism with autonomy at the top of the "moral mountain." Lay people may differ in the way they invoke and balance the various ethical interests. We explored lay people's ethical attitudes to organ donation. METHODS: Respondents (n=196) ranked 42 opinion-statements on organ donation according to a 9-category symmetrical distribution. Statements' scores were analyzed by averaging-analysis and Q-methodology. RESULTS: Respondents' mean (SD) age was 34.5 (10.6) years, 53% were women, 69% Muslims (30% Christians), 29% Saudis (26% Filipinos), and 38% healthcare-related. The most-agreeable statements were "Acceptable if benefit to recipient large," "Explicit donor consent and family approval for live donation," "Acceptable if directed to family member," and "Explicit donor consent and family approval for postmortem donation." The most-disagreeable statements were "Donor consent and family approval not required for postmortem donation," "Acceptable with purely materialistic motivation," and "Only donor no-known objection for postmortem donation." Women, Christians, and healthcare respondents gave higher rank to "Explicit donor consent and family approval for live donation," "Only donor family consent required for postmortem donation," and "Acceptable if organ distribution equitable," respectively, and Muslims gave more weight to donor/family harm (p ≤0.001). Q-methodology identified various ethical resolution models that were associated with religious affiliation and included relatively "motives-concerned," "family-benefit-concerned," "familism-oriented," and "religious or non-religious altruism-concerned" models. Of 23 neutral statements on averaging-analysis, 48% and 65% received extreme ranks in ≥1 women and men Q-methodology models, respectively. CONCLUSION: 1) On average, recipient benefit, requirement of both explicit donor consent and family approval, donor-recipient relationship, and motives were predominant considerations; 2) ranking of some statements was associated with respondents' demographics; 3) Q-methodology identified various ethical resolution models that were partially masked by averaging-analysis; and 4) strong virtue and familism approaches in our respondents provide some empirical evidence against principlism adequacy.

Circumferential Negative Intermittent Pressure to the Midarm Does Not Impair Digital O2 Saturation: A Randomized Controlled Study.

BACKGROUND: Circumferential negative pressure wound therapy is commonly used to manage wounds and enhance the healing process. A theoretical concern was recently raised that circumferential negative pressure wound therapy may have a negative effect on perfusion distally. METHODS: In a randomized study, we applied circumferential negative pressure (125 mm Hg) to the midarm of 13 healthy volunteers through InfoV.A.C. Therapy Unit device. The pressure was applied intermittently (5 minutes on and 2 minutes off) for 9 hours. The same device without negative pressure was applied to the contralateral midarm as control. Bilateral index finger O2 saturation (SpO2) was measured every 30 minutes using digital pulse oximetry. RESULTS: Mean (SD) age of the volunteers was 32.2 (9.5) years, and 61.5% were male. Mean (SD) area under the curve from time 0 to 9 hours of SpO2 was 890.56 (6.69) and 889.71 (6.23) %xh in the intervention and control arms, respectively (P = 0.35). O2 saturation was ≥94% at all observation times in both arms, and no adverse events were identified. CONCLUSIONS: Circumferential negative intermittent pressure of 125 mm Hg applied to the midarm of healthy volunteers for 9 hours does not adversely affect digital SpO2.

Superfluous amylase/lipase testing at a tertiary care hospital: a retrospective study.

BACKGROUND: Measuring both serum amylase and lipase in the setting of acute pancreatitis is not recommended and monitoring changes in amylase and lipase levels after diagnostic results is of little added value. The extent of the two types of superfluous amylase/lipase testing at our institution is unknown. OBJECTIVE: Explore the extent of superfluous amylase/lipase testing. DESIGN: Medical record review. SETTINGS: Tertiary care, teaching hospital. PATIENTS AND METHODS: We retrospectively reviewed all amylase and lipase tests performed over a recent 12-month period. Amylase tests were considered superfluous if they were ordered with lipase tests at the same time or if they were repeated after diagnostic amylase results. They were considered questionably superfluous if they were repeated alone after non-diagnostic amylase results. Lipase tests were considered superfluous if they were repeated after diagnostic lipase results and questionably superfluous if they were repeated after non-diagnostic lipase results. MAIN OUTCOME MEASURES: Number and percentage of lipase and amylase tests that were superfluous or questionably superfluous. SAMPLE SIZE: 23 950. RESULTS: Superfluous testing was identified in 30.6% of 23 950 amylase/lipase tests and questionably superfluous testing in 12.4%. Of the 7330 superfluous tests, 94.8% were due to simultaneous amylase/lipase testing and 5.2% to repeated lipase testing after diagnostic results. The rate of superfluous and questionably superfluous testing was significantly higher in the inpatient setting compared to emergency department or outpatient settings ( P<.0001). Of the 6483 amylase tests obtained simultaneously with non-diagnostic lipase tests, only 36 (0.6%) showed a diagnostic result. Furthermore, only 0.7% and 3.6% of amylase tests that were repeated after normal and borderline results, respectively, were diagnostic and 1.1% and 9.3% of lipase tests that were repeated after normal and borderline results, respectively, were diagnostic. CONCLUSIONS: About one third of amylase/lipase testing appears to be superfluous, mainly due to simultaneous amylase/lipase testing. Since only 0.6% of simultaneous amylase/lipase tests showed diagnostic amylase with non-diagnostic lipase levels, quality improvement initiatives should be directed at reducing this low-value practice. Repeating amylase/lipase tests following normal results is of little value. LIMITATIONS: Clinical notes and imaging studies were not reviewed. CONFLICT OF INTEREST: None.

Lay People's Ethical Attitudes To Placebo Treatment: A Q-Methodology Study.

BACKGROUND: Placebo-treatment acceptability is debated among ethicists, mostly due to conflict between respect-to-autonomy and beneficence principles. It is not clear how lay people balance these and other ethical principles. METHODS: One hundred and eighty-seven respondents rank-ordered 42 opinion statements covering various ethical aspects of placebo-treatment, according to a 9-category symmetrical distribution. We analyzed statements' scores using averaging-analysis and by-person factor analysis (Q-methodology). RESULTS: Respondents' mean (SD) age was 34.6 (10.6) years, 54% were women, 40% healthcare-related, 68% Muslims (31% Christians), and 39% received general education in Saudi Arabia (24% in the Philippines). On averaging-analysis, the most-agreeable statements were "Acceptable if benefit to patient large" and "Acceptable with physician intent to benefit patient". The most-disagreeable statements were "Acceptable with physician self-benefit intent" and "Acceptable with large harm to other patients". Muslims gave a higher rank to "Giving no description is acceptable", "Acceptable with small benefit to patient", and "Acceptable with physician intent to benefit patient" and a lower rank to "Acceptable to describe as inactive drug", "Acceptable with physician intent to please patient caring relative", and "Acceptable with moderate harm to other patients" (p<0.01). Q-methodology detected several ethical attitude models that were mostly multi-principled and consequentialism-dominated. The majority of Christian and Philippines-educated women loaded on a "relatively family and deception-concerned" model, whereas the majority of Muslim and Saudi Arabia-educated women loaded on a "relatively common-good-concerned" model. The majority of Christian and healthcare men loaded on a "relatively deception-concerned" model, whereas the majority of Muslim and non-healthcare men loaded on a "relatively motives-concerned" model. Of nine intent-related statements, ≥2 received extreme rank on averaging-analysis and in 100% of women and men models. CONCLUSION: 1) On averaging-analysis, patient's beneficence (consequentialism) followed by physician's intent (virtue ethics) were more important than deception (respect-to-autonomy). 2) Q-methodology identified several ethical attitude models that were mostly multi-principled and associated with respondents' demographics.

Prediction of life-story narrative for end-of-life surrogate's decision-making is inadequate: a Q-methodology study.

BACKGROUND: Substituted judgment assumes adequate knowledge of patient's mind-set. However, surrogates' prediction of individual healthcare decisions is often inadequate and may be based on shared background rather than patient-specific knowledge. It is not known whether surrogate's prediction of patient's integrative life-story narrative is better. METHODS: Respondents in 90 family pairs (30 husband-wife, 30 parent-child, 30 sibling-sibling) rank-ordered 47 end-of-life statements as life-story narrative measure (Q-sort) and completed instruments on decision-control preference and healthcare-outcomes acceptability as control measures, from respondent's view (respondent-personal) and predicted pair's view (respondent-surrogate). They also scored their confidence in surrogate's decision-making (0 to 4 = maximum) and familiarity with pair's healthcare-preferences (1 to 4 = maximum). Life-story narratives' prediction was examined by calculating correlation of statements' ranking scores between respondent-personal and respondent-surrogate Q-sorts (projection) and between respondent-surrogate and pair-personal Q-sorts before (simulation) and after controlling for correlation with respondent-personal scores (adjusted-simulation), and by comparing percentages of respondent-surrogate Q-sorts co-loading with pair-personal vs. respondent-personal Q-sorts. Accuracy in predicting decision-control preference and healthcare-outcomes acceptability was determined by percent concordance. Results were compared among subgroups defined by intra-pair relationship, surrogate's decision-making confidence, and healthcare-preferences familiarity. RESULTS: Mean (SD) age was 35.4 (10.3) years, 69% were females, and 73 and 80% reported ≥ very good health and life-quality, respectively. Mean surrogate's decision-making confidence score was 3.35 (0.58) and 75% were ≥ familiar with pair's healthcare-preferences. Mean (95% confidence interval) projection, simulation, and adjusted-simulation correlations were 0.68 (0.67-0.69), 0.42 (0.40-0.44), and 0.26 (0.24-0.28), respectively. Out of 180 respondent-surrogate Q-sorts, 24, 9, and 32% co-loaded with respondent-personal, pair-personal, or both Q-sorts, respectively. Accuracy in predicting decision-control preference and healthcare-outcomes acceptability was 47 and 52%, respectively. Surrogate's decision-making confidence score correlated with adjusted-simulation's correlation score (rho = 0.18, p = 0.01). There were significant differences among the husband-wife, parent-child, and sibling-sibling subgroups in percentage of respondent-surrogate Q-sorts co-loading with pair-personal Q-sorts (38, 32, 55%, respectively, p = 0.03) and percent agreement on healthcare-outcomes acceptability (55, 35, and 67%, respectively, p = 0.002). CONCLUSIONS: Despite high self-reported surrogate's decision-making confidence and healthcare-preferences familiarity, family surrogates are variably inadequate in simulating life-story narratives. Simulation accuracy may not follow the next-of-kin concept and is 38% based on shared background.

A Simple Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Measurement of Cortisol Level in Human Saliva.

A simple ultraperformance liquid chromatography-tandem mass spectrometry assay for measurement of cortisol level in human saliva was developed and validated. Saliva samples containing cortisol were spiked with tolperisone as internal standard (IS) and extracted with a mixture of methyl tert-butyl ether and hexane (8:2, v:v). After solvent evaporation, residue was reconstituted in 100 µl mobile phase. Analysis was performed on Atlantis dC18 column (2.1 × 100 mm, 3 µm particle size) with a mobile phase composed of acetonitrile and 2 mM ammonium acetate (50:50, v:v) and delivered at a flow rate of 0.3 ml/minute. Mass spectrometry acquisition was performed with multiple reaction monitoring in positive-ion mode for cortisol and IS (m/z: 363.1 → 121.0 and 246.0 → 97.9, respectively). Retention times of cortisol and IS were about 1.35 and 2.45 minutes, respectively. The relationship between cortisol level and peak area ratio of cortisol to IS was linear in the range of 0.5-100 ng/ml. Intra- and interday coefficient of variation and bias were ≤ 9.0% and ≤12.0%, respectively. Mean extraction recoveries of cortisol and IS from saliva samples were 92% and 94%, respectively. Using the method, cortisol was found to be ≥ 86% stable in processed (24 hours at room temperature or 48 hours at -20°C) and ≥ 91% stable in unprocessed (24 hours at room temperature or 20 weeks at -20°C) saliva samples. Further, the method was successfully applied to determine daily cortisol profile in saliva samples of a healthy volunteer.

Vitamin-D2 treatment-associated decrease in 25(OH)D3 level is a reciprocal phenomenon: a randomized controlled trial.

BACKGROUND: Vitamin-D2 (D2) treatment has been associated with a decrease in 25-hydroxy (25(OH)) vitamin-D3 (D3) level, suggesting that D3 treatment would be preferred to raise total 25(OH) vitamin-D (D) level. We postulated that D2 treatment-associated decrease in 25(OH)D3 level is related to the increase in 25(OH)D level rather than being D2-specific, and thus there would be a similar D3 treatment-associated decrease in 25(OH)D2 level. METHODS: Fifty volunteers were block-randomized to 50,000 IU D2 or placebo orally once (study-1) and fifty volunteers received 50,000 IU D2 orally once and 4 days later block-randomized to 50,000 IU D3 or placebo orally once (study-2). Interventions were concealed from volunteers and research coordinators and blindly-administered. Serum 25(OH)D2 and 25(OH)D3 levels were blindly-determined at baseline and days 14, 28, 42, and 56, post-randomization by high performance liquid chromatography assay. Results of 97 participants were analyzed. Primary outcome measure was day-28 D2-associated change in 25(OH)D3 level in study-1 and D3-associated change in 25(OH)D2 level in study-2, adjusted for baseline levels. RESULTS: Mean (95% confidence interval) difference between the active and placebo arms in the decrease in day-28 25(OH)D3 (study-1) and 25(OH)D2 (study-2) levels was 13.2 (9.7 to 16.6) and 9.8 (5.2 to 14.4) nmol/L, respectively. Corresponding differences at day-56 were 10.8 (6.8 to 14.8) and 1.7 (- 7.6 to 11.1) nmol/L, respectively. The difference between the placebo and active arms in area-under-the-curve at day-28 (AUC28) and day-56 (AUC56) were 262.3 (197.8 to 326.7) and 605.1 (446.3 to 784.0) for 25(OH)D3 (study-1) and 282.2 (111.2 to 453.3) and 431.2 (179.3 to 683.2) nmol.d/L for 25(OH)D2 (study-2), respectively. There were significant correlations between day-28 changes in 25(OH)D2 and 25(OH)D3 levels in study-1 (rho = - 0.79, p < 0.001) and study-2 (rho = - 0.36, p = 0.01), and between day-28 changes in 25(OH)D2 level and baseline 25(OH)D level in study-2 (rho = - 0.42, p = 0.003). CONCLUSIONS: Compared to placebo, D3 treatment is associated with a decrease in 25(OH)D2 level similar in magnitude to D2-treatment associated decrease in 25(OH)D3 level; however, the D3-placebo difference in 25(OH)D2 level is shorter-lasting. Changes in 25(OH)D2 and 25(OH)D3 levels are correlated with each other and with baseline 25 (OH) D levels, suggesting a common regulatory mechanism. TRIAL REGISTRATION: ClinicalTrial.gov identifier: NCT03035084 (registered January 27, 2017).

Evaluation of Omeprazole Limited Sampling Strategies to Estimate Constitutive Cytochrome P450 2C19 Activity in Healthy Adults.

BACKGROUND: Limited sampling strategy (LSS) is a validated method to estimate pharmacokinetic (PK) parameters from a reduced number of samples. Omeprazole is used to phenotype in vivo cytochrome P450 (CYP) 2C19 activity. This study examined an LSS using 2 estimation methods to determine apparent oral clearance (CL/F) and thus CYP2C19 activity. METHODS: Data from 7 previously published studies included healthy subjects receiving a single, oral dose of omeprazole with intensive PK sampling. CL/F was estimated using noncompartmental analysis (NCA) and population PK modeling. LSS was simulated by selecting the 1, 2, 4, and/or 6-hour postdose time points. Linear regression was performed to assess whether CL/F estimated from limited sampling could accurately predict CL/F from the full PK profile. RESULTS: Median CL/F was 23.7 L/h by NCA and 19.3 L/h by population PK modeling. In comparing the LSS NCA estimated versus observed CL/F, all evaluated linear regression models had unacceptable coefficients of determination (r, range: 0.14-0.81). With the population PK approach, 737 plasma concentrations (n = 71) and CYP2C19 genotype data were described with a 1-compartment structural model with mixed zero and first-order absorption and lag time. In comparing the population PK LSS estimated versus observed CL/F, all evaluated linear regression models had unacceptable r (range: 0.02-0.74). Post hoc comparison of CYP2C19 poor metabolizers versus CYP2C19 extensive metabolizers resulted in significantly lower CL/F in poor metabolizers versus extensive metabolizers. CONCLUSIONS: Omeprazole LSS performed poorly in estimating CL/F using 2 separate estimation approaches and does not seem to be a suitable method for determining CYP2C19 activity.

Generic-reference and generic-generic bioequivalence of forty-two, randomly-selected, on-market generic products of fourteen immediate-release oral drugs.

BACKGROUND: The extents of generic-reference and generic-generic average bioequivalence and intra-subject variation of on-market drug products have not been prospectively studied on a large scale. METHODS: We assessed bioequivalence of 42 generic products of 14 immediate-release oral drugs with the highest number of generic products on the Saudi market. We conducted 14 four-sequence, randomized, crossover studies on the reference and three randomly-selected generic products of amlodipine, amoxicillin, atenolol, cephalexin, ciprofloxacin, clarithromycin, diclofenac, ibuprofen, fluconazole, metformin, metronidazole, paracetamol, omeprazole, and ranitidine. Geometric mean ratios of maximum concentration (Cmax) and area-under-the-concentration-time-curve, to last measured concentration (AUCT), extrapolated to infinity (AUCI), or truncated to Cmax time of reference product (AUCReftmax) were calculated using non-compartmental method and their 90% confidence intervals (CI) were compared to the 80.00%-125.00% bioequivalence range. Percentages of individual ratios falling outside the ±25% range were also determined. RESULTS: Mean (SD) age and body-mass-index of 700 healthy volunteers (28-80/study) were 32.2 (6.2) years and 24.4 (3.2) kg/m2, respectively. In 42 generic-reference comparisons, 100% of AUCT and AUCI CIs showed bioequivalence, 9.5% of Cmax CIs barely failed to show bioequivalence, and 66.7% of AUCReftmax CIs failed to show bioequivalence/showed bioinequivalence. Adjusting for 6 comparisons, 2.4% of AUCT and AUCI CIs and 21.4% of Cmax CIs failed to show bioequivalence. In 42 generic-generic comparisons, 2.4% of AUCT, AUCI, and Cmax CIs failed to show bioequivalence, and 66.7% of AUCReftmax CIs failed to show bioequivalence/showed bioinequivalence. Adjusting for 6 comparisons, 2.4% of AUCT and AUCI CIs and 14.3% of Cmax CIs failed to show bioequivalence. Average geometric mean ratio deviation from 100% was ≤3.2 and ≤5.4 percentage points for AUCI and Cmax, respectively, in both generic-reference and generic-generic comparisons. Individual generic/reference and generic/generic ratios, respectively, were within the ±25% range in >75% of individuals in 79% and 71% of the 14 drugs for AUCT and 36% and 29% for Cmax. CONCLUSIONS: On-market generic drug products continue to be reference-bioequivalent and are bioequivalent to each other based on AUCT, AUCI, and Cmax but not AUCReftmax. Average deviation of geometric mean ratios and intra-subject variations are similar between reference-generic and generic-generic comparisons. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01344070 (registered April 3, 2011).

Large Intra-subject Variability in Caffeine Pharmacokinetics: Randomized Cross-over Study of Single Caffeine Product.

Background Average bioequivalence has been criticized for not adequately addressing individual variations. Importance of subjects' blinding in bioequivalence studies has not been well studied. We explored the extent of intra-subject pharmacokinetic variability and effect of drug-ingestion unawareness in subjects taking single caffeine product. Methods A single-dose randomized cross-over design was used to compare pharmacokinetics of 200 mg caffeine, described as caffeine (overt) or as placebo (covert). Maximum concentration (Cmax), Cmax first time (Tmax), area-under-the-concentration-time-curve, to last measured concentration (AUCT), extrapolated to infinity (AUCI), or to Tmax of overt caffeine (AUCOverttmax), and Cmax/AUCI were calculated blindly using standard non-compartmental method. Percentages of individual covert/overt ratios that are outside the ±25% range were determined. Covert-vs-overt effect on caffeine pharmacokinetics was evaluated by 90% confidence interval (CI) and 80.00-125.00% bioequivalence range. Results 32 healthy subjects (6% females, mean (SD) age 33.3 (7.2) year) participated in the study (28 analysed). Out of the 28 individual covert/overt ratios, 23% were outside the ±25% range for AUCT, 30% for AUCI, 20% for AUCOverttmax, 30% for Cmax, and 43% for Tmax. There was no significant covert-vs-overt difference in any of the pharmacokinetic parameters studied. Further, the 90% CIs for AUCT, AUCI, Cmax, AUCOverttmax, and Cmax/AUCI were all within the 80.00-125.00% bioequivalence range with mean absolute deviation of covert/overt ratios of 3.31%, 6.29%, 1.43%, 1.87%, and 5.19%, respectively. Conclusions Large intra-subject variability in main caffeine pharmacokinetic parameters was noted when comparing an oral caffeine product to itself. Subjects' blinding may not be important in average bioequivalence studies.

Does the placebo effect modulate drug bioavailability? Randomized cross-over studies of three drugs.

BACKGROUND: Medication effect is the sum of its drug, placebo, and drug*placebo interaction effects. It is conceivable that the interaction effect involves modulating drug bioavailability; it was previously observed that being aware of caffeine ingestion may prolong caffeine plasma half-life. This study was set to evaluate such concept using different drugs. METHODS: Balanced single-dose, two-period, two-group, cross-over design was used to compare the pharmacokinetics of oral cephalexin, ibuprofen, and paracetamol, each described by its name (overt) or as placebo (covert). Volunteers and study coordinators were deceived as to study aim. Drug concentrations were determined blindly by in-house, high performance liquid chromatography assays. Terminal-elimination half-life (t½) (primary outcome), maximum concentration (Cmax), Cmax first time (Tmax), terminal-elimination-rate constant (λ), area-under-the-concentration-time-curve, to last measured concentration (AUCT), extrapolated to infinity (AUCI), or to Tmax of overt drug (AUCOverttmax), and Cmax/AUCI were calculated blindly using standard non-compartmental method. Covert-vs-overt effect on drug pharmacokinetics was evaluated by analysis-of-variance (ANOVA, primary analysis), 90% confidence interval (CI) using the 80.00-125.00% bioequivalence range, and percentage of individual pharmacokinetic covert/overt ratios that are outside the +25% range. RESULTS: Fifty, 30, and 50 healthy volunteers (18%, 10%, and 6% females, mean (SD) age 30.8 (6.2), 31.4 (6.6), and 31.2 (5.4) years) participated in 3 studies on cephalexin, ibuprofen, and paracetamol, respectively. Withdrawal rate was 4%, 0%, and 4%, respectively. Eighteen blood samples were obtained over 6, 10, and 14 h in each study period of the three drugs, respectively. ANOVA showed no significant difference in any pharmacokinetic parameter for any of the drugs. The 90% CIs for AUCT, AUCI, Cmax, AUCOverttmax, and Cmax/AUCI were within the bioequivalence range, except for ibuprofen Cmax (76.66-98.99), ibuprofen Cmax/AUCI (77.19-98.39), and ibuprofen (45.32-91.62) and paracetamol (51.45-98.96) AUCOverttmax. Out of the 126 individual covert/overt ratios, 2.0-16.7% were outside the +25% range for AUCT, 2.0-4.2% for AUCI, 25.0-44.9% for Cmax, 67.3-76.7% for AUCOverttmax, and 45.8-71.4% for Tmax. CONCLUSIONS: This study couldn't confirm that awareness of drug ingestion modulates its bioavailability. However, it demonstrates the trivial effect of blinding in bioequivalence studies and the extent of bio-variability that would be expected when comparing a drug product to itself. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01501747 (registered Dec 26, 2011).

Differential effects of vitamin D2 and D3 supplements on 25-hydroxyvitamin D level are dose, sex, and time dependent: a randomized controlled trial.

BACKGROUND: Vitamin D (D) supplements are indispensable for its world-wide deficiency. Controversy continues on ergocalciferol (D2) and cholecalciferol (D3) relative potency as well as on dosing-schedule and sex role in raising 25-hydroxy D (25(OH)D) level, the best indicator of D status. METHODS: We randomized 279 adults to daily D2, D3, D2/D3, or placebo; 2-weekly D2 or D3; or 4-weekly D2 or D3 (250,000 IU over/140 days). Randomization sequence, stratified by body-mass-index (BMI) and sex, was concealed from study coordinators and participants who were then blinded to capsules' content. D2, D3, 25(OH)D2, and 25(OH)D3 Serum levels were determined blindly on days 0,1,2,3,4,7,14, and 2-weekly thereafter by high performance liquid chromatography assay. The results of 269 participants were available for analysis. Primary endpoint was area-under-the-curve (AUC) of 25(OH)D (25(OH)D2 + 25(OH)D3) adjusted for sex, BMI, and baseline 25(OH)D level. RESULTS: Mean(SD) age was 33.0(8.5) year, 41% were males, and 85% completed follow-up. Baseline 25(OH)D level was 39.8(11.9) and increased by 3.3(11.6) and 28.6(16.3) nmol/L, in the placebo and active-treatment groups, respectively. AUC from day 0 to 140 (AUC140) of 25(OH)D was 40% (D3 daily) to 55% (D3 2-weekly) higher with active-treatment than placebo (p < 0.001). 25(OH)D2 AUC140 was higher in daily than 2-weekly (17%, p = 0.006) and 4-weekly (20%, p = 0.001) D2-treated groups. 25(OH)D3 AUC140 was lower in daily than 2-weekly (11%, p = 0.002) and 4-weekly D3-treated groups (10%, p = 0.008). In D2-treated groups, there was 16.4 nmol/L decrease in 25(OH)D3 level that correlated (p < 0.001) with 25(OH)D2 level increase (r = 0.48) and baseline 25(OH)D level (r = 0.58), in one participant with measurable baseline 25(OH)D2 level, D3 caused a similar decrease in 25(OH)D2 level, while in the D2/D3-treated group, 25(OH)D3 level didn't increase. Incremental AUC from day 0 to 7 (AUC7) of D3 and 25(OH)D3 in D3-treated groups were 118-243% higher and 31-39% lower, respectively, than incremental AUC7 of D2 and 25(OH)D2 in D2-treated groups. Incremental AUC7 of D3 and 25(OH)D3 in D3-treated groups and D2 and 25(OH)D2 in D2-treated groups were higher in females than males (55, 13, 64, and 28%, respectively). Baseline 25(OH)D level predicted response to D2 and D3 (p < 0.001), whereas, BMI was significant predictor only for early response to D2. CONCLUSIONS: Effects of D2 and D3 supplements on 25 (OH)D level may be dosing-schedule and sex-dependent. D2-associated reduction in 25(OH)D3 level may be related to total 25(OH)D level rather than being D2-specific. D2 may be 25-hydroxylated faster than D3. TRIAL REGISTRATION: ClinicalTrial.gov identifier: NCT01170494 (registered July 25, 2010).