Dose Uniformity of Topical Corticosteroids: A Simulated Trial of Fluorometholone Acetate 0.1% and Loteprednol Etabonate Gel 0.5.

PURPOSE: The purpose of the study was to determine the concentrations of Flarex® and Lotemax® when shaken and not shaken. Many patients fail to shake or inappropriately shake suspensions of corticosteroids before instillation as directed. This study was designed to help determine what concentration of corticosteroid these patients are receiving. In addition, independent confirmation of loteprednol etabonate ophthalmic gel dose uniformity was determined and compared as a possible alternative. METHODS: Drug concentrations of shaken versus unshaken Flarex and Lotemax were determined over a 20-day simulated tapered course in our institutional laboratory. Collected samples were analyzed by reversed-phase high-performance liquid chromatography with photodiode array detection at 240 nm. RESULTS: Flarex had a mean concentration of 93.7% of the declared concentration when shaken and 7.25% when not shaken. The difference between these groups was statistically significant (P = 0.0001). Lotemax had a mean concentration of 96.74% of the declared concentration when shaken and a mean concentration of 98.97% when not shaken. The difference between these groups was not statistically significant (P = 0.194). CONCLUSIONS: Flarex maintains dose uniformity when shaken. When not shaken, it has poor dose uniformity. Lotemax was consistent whether shaken or not in our study and can be considered to eliminate the variability of poor patient compliance with shaking. The manufacturers of both drugs recommend shaking before application.

Characterization of the Fluorescent Spectra and Intensity of Dabigatran and Dabigatran Etexilate: Application to HPLC Analysis with Fluorescent Detection†.

There is considerable interest in dabigatran etexilate (Pradaxa) and its major metabolite, dabigatran, which has been shown to be an important inhibitor of thrombin and clotting. In this study, the fluorescent excitation and emission spectra of dabigatran and dabigatran etexilate were characterized. In addition, a ultra performance liquid chromatography (UPLC) and high performance liquid chromatography (HPLC) method using fluorescent detection was developed for the analysis of dabigatran. Dabigatran and dabigatran etexilate were found to have excitation and emission maxima of 310 and 375 nm and 335 and 400 nm, respectively. UPLC analysis of dabigatran standards and plasma dabigatran samples were analyzed on a reversed phase C-18 column with methanol-water (70:30, v/v) as the mobile phase. The lower limit of quantitation for dabigatran was 10.0 ng/mL for both the standards and plasma samples. Standard curves were linear from 10.0 to 1000.0 ng/mL (R2 = 0.995). Within-day coefficient of variations of the fluorometric method at 50.0, 100.0 and 500.0 ng/mL were 1.38%, 4.83% and 2.31%, respectively. The intense fluorescent properties of dabigatran permit the sensitive and specific UPLC or HPLC fluorescent analysis of dabigatran.

High-Performance Liquid-Chromatographic Analysis of Plasma Iohexol Concentrations.

In this study, a high-performance liquid-chromatographic (HPLC) method using photodiode array detection and isocratic conditions was developed for the analysis of plasma iohexol concentrations. Plasma proteins were precipitated with 1:1 volume of plasma and acetonitrile-ethanol-water (60:38.4:1.6, v/v/v). Iohexol concentrations in the supernatant phase were analyzed on a Waters Symmetry C-18 reversed-phase column under isocratic conditions at 245 nm. The extraction recoveries of iohexol from plasma were >95% and the plasma iohexol calibration curves were linear (R(2) ≥ 0.9998) from 10 to 1500 µg/mL. The within-day coefficients of variation (CVs) at plasma iohexol concentrations of 100, 375, 750 and 1500 µg/mL were 5.1, 3.5, 1.3 and 2.5%, respectively; the between-day CVs at 100, 375, 750 and 1500 µg/mL were 8.6, 4.2, 4.0 and 3.7%, respectively. The day-to-day accuracies of the method at plasma iohexol concentrations of 50, 100, 375, 750 and 1500 µg/mL were 89.0, 99.4, 108.4, 103.6 and 101.2%, respectively (n = 5). The lower limit of plasma iohexol quantitation was 10 µg/mL and no interferences >9 µg/mL were found in over 75 pre-dose porcine plasma samples. The applicability of the method was demonstrated by determining the glomerular filtration rates of iohexol in the porcine (Sus scrofa) model.

Characterization of the Fluorescent Spectra and Intensities of Various Lignans: Application to HPLC Analysis with Fluorescent Detection†.

There is considerable interest in dietary lignans since they have been shown to have antioxidant, estrogenic and lipid-lowering activity in humans. In this study, the fluorescent excitation and emission spectra of seven lignans were characterized and their relative fluorescent intensities compared. The lignans were found to have similar excitation (286.6 ± 2.5 nm, X ± SD) and emission (320.1 ± 6.4 nm) maxima; however, their fluorescence intensities on a molar basis decreased in the following order: asarinin, sesamin, sesamolin, seco-isolariciresinol, seco-isolariciresinol diglucoside and matairesinol. Enterolactone, a mammalian lignan conversion product, and sesamol, an antioxidant found in sesame oil, also exhibited significant fluorescence excitation and emission intensities. A high-performance liquid chromatographic method using photodiode array (PDA) and fluorescent detection was developed for the analysis of the individual lignans. Analysis was performed on a reversed phase C-18 column with methanol-water (70:30, v/v) as the mobile phase. With fluorescent detection, the limits of quantitation (LOQ) was 0.1 ng or 2.82 nmol for sesamin and asarinin, 2.70 nmol for sesamolin, 2.76 nmol for seco-isolariciresinol, 1.45 nmol for seco-isolariciresinol diglucoside, 2.79 nmol for matairesinol and 0.5 ng or 1.67 nmol for enterolactone. With PDA detection, the LOQ was a 1000-fold less sensitive than with fluorescent detection.

Liquid chromatographic mass spectrometric (LC/MS/MS) determination of plasma hydroxocobalamin and cyanocobalamin concentrations after hydroxocobalamin antidote treatment for cyanide poisoning.

Cyanide poisoning occurs in individuals after fire smoke inhalation and after oral ingestion of cyanide. Hydroxocobalamin (HOCbl), a hydroxylated form of vitamin B(12), is often used as an antidote to treat cyanide toxicity. It has a high affinity for cyanide and rapidly removes cyanide from tissue by forming cyanocobalamin (CNCbl). Little information is available on the pharmacokinetics of HOCbl and CNCbl largely because of the lack of analytical methods for analyzing HOCbl and CNCbl. In this study, we developed a new liquid chromatographic mass spectrometric (LC/MS/MS) method for the quantitative analysis of plasma HOCbl and CNCbl in the porcine (Sus scrofa) model. The method uses on-column extraction, reversed phase gradient chromatography, and multiple reaction monitoring (MRM) for quantitation. MRM transitions monitored were 664.7→147.3 and 664.7→359.2 for HOCbl and 678.8→147.3, 678.8→359.1 678.8→457.1 for CNCbl. The limit of detection (LOD) and the lower limit of quantitation (LLOQ) were 1.0 and 1.0 µmole/L, respectively, for plasma HOCbl and 0.1 and 0.5 µmole/L for plasma CNCbl. The within-day and between-day CVs were 4.3 and 6.4% for plasma HOCbl at 500.0 µmole/L and 5.5 and 5.7% for CNCbl at 100.0 µmole/L (n=6). The plasma HOCbl and CNCbl calibrations curves were linear from 100.0 to 2000.0 and 50.0 to 500.0 µmole/L, respectively. Based on 6 separate calibration curves the average linear regression coefficient (R(2)) for both HOCbl and CNCbl was 0.992. The LC/M/MS method was found to be accurate and precise and has been validated by determining the plasma HOCbl and CNCbl concentrations in 11 pigs that were treated with HOCbl for cyanide poisoning.

Association of serum bilirubin and promoter variations in HMOX1 and UGT1A1 genes with sporadic colorectal cancer.

Heme oxygenase-1 (HMOX1) and bilirubin UDP-glucuronosyltransferase (UGT1A1) enzymes, both involved in bilirubin homeostasis, play an important role in the oxidative stress defense. The objective of our study was to assess the effect of promoter variations of HMOX1 and UGT1A1 genes and of serum bilirubin on the risk of sporadic colorectal cancer (CRC). This exploratory case-control study was based on 777 CRC patients and 986 controls from the Czech Republic. The (GT)(n) and (TA)(n) dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. In addition, the A(-413)T variant in HMOX1 promoter was also analyzed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Serum bilirubin levels were compared in a subset of 174 cases and 247 controls, for whom biochemical data were available. After adjustment for age, a significant association between CRC risk and UGT1A1*28 allele carrier status was detected [odds ratio (95% confidence intervals) = 0.80 (0.60-0.97), p = 0.022]. No association between CRC risk and individual HMOX1 gene variants was observed, although a diplotype analysis revealed an increased risk for a specific HMOX1 genotype combination. These effects were more pronounced in males. Substantially lower serum bilirubin levels were detected in CRC patients compared to the controls (p < 0.001); each 1 µmol/L decrease in serum bilirubin was associated with a 7% increase of CRC risk (p < 0.001). In conclusion, UGT1A1*28 allele carrier status might be a protective factor against the development of CRC in the male population, whereas low serum bilirubin levels are associated with an increased risk of CRC in both genders.

Serum bilirubin and genes controlling bilirubin concentrations as biomarkers for cardiovascular disease.

BACKGROUND: Serum bilirubin has been consistently shown to be inversely related to cardiovascular disease (CVD). Recent studies showed serum bilirubin to be associated with CVD-related factors such as diabetes, metabolic syndrome, and body mass index. Although the association of serum bilirubin with CVD has been found in both retrospective and prospective studies, less information is available on the role of genes that control bilirubin concentrations and their association with CVD. CONTENT: In this review, we provide detailed information on the identity of the major genes that control bilirubin concentrations and their association with serum bilirubin concentrations and CVD risk. We also update the results of the major studies that have been performed on the association between serum bilirubin, CVD, and CVD-related diseases such as diabetes or metabolic syndrome. Studies consistently indicate that bilirubin concentrations are inversely associated with different types of CVD and CVD-related diseases. A conditional linkage study indicates that UGT1A1 is the major gene controlling serum bilirubin concentrations, and this finding has been confirmed in recent genomewide association studies. Studies also indicate that individuals homozygous for UGT1A1*28 have a significantly lower risk of developing CVD than carriers of the wild-type alleles. SUMMARY: Serum bilirubin has a protective effect on CVD and CVD-related diseases, and UGT1A1 is the major gene controlling serum bilirubin concentrations. Pharmacologic, nonpharmacologic, or genetic interventions that increase serum bilirubin concentrations could provide more direct evidence on the role of bilirubin in CVD prevention.

Predicting irreparable renal ischemic injury using a real-time marker in the porcine model.

PURPOSE: We determined the maximal renal tolerance of warm ischemia using renal cortical interstitial metabolic changes to identify a potential real-time marker of irreparable renal function. MATERIALS AND METHODS: Using a single kidney model 3 groups of 5 pigs each underwent 120, 150 and 180 minutes of warm ischemia, respectively. Microdialysis samples were collected before, during and after ischemia. Renal function assessments consisting of serum creatinine and GFR measurements were performed before ischemia and on post-ischemia days 1, 5, 9, 14 and 28. Kidneys exposed and not exposed to ischemia were collected for histological study. RESULTS: Interstitial glucose and pyruvate concentrations decreased, while lactate concentrations increased to stable levels during ischemia. Glutamate spiked at 30 minutes of ischemia and subsequently tapered, while glycerol increased throughout warm ischemia time. At post-ischemia day 28 renal function returned to pre-ischemia baseline levels in the group with 120 minutes of ischemia but did not recover to baseline in the 150 and 180-minute ischemic groups. Functional data correlated with histological findings. The 120-minute maximal renal tolerance of warm ischemia correlated with a mean +/- SD glycerol concentration of 167 +/- 24 micromol/l. CONCLUSIONS: Interstitial glycerol is a real-time, renal unit specific, minimally invasive marker of renal function deterioration. Exposure of porcine kidneys to ischemic insults resulting in renal cortical interstitial glycerol concentrations higher than 167 micromol/l is associated with irreparable functional damage in this model.

Gilbert syndrome, UGT1A1*28 allele, and cardiovascular disease risk: possible protective effects and therapeutic applications of bilirubin.

Serum bilirubin has been shown to be inversely related to cardiovascular disease (CVD) in both retrospective and prospective studies. Meta-analysis of existing studies has also confirmed that serum bilirubin concentrations are inversely related to CVD. Less information is known about the protective effects of slightly elevated serum bilirubin concentrations. In this review, we will focus primarily on the association of serum bilirubin and CVD and the possible protective roles of bilirubin, heme oxygenase (HO), and bilirubin UDP-glucuronosyltransferase (UGT1A1). HO and biliverdin reductase control the formation of bilirubin, whereas UGT1A1 controls bilirubin conjugation and clearance. Because of the health and therapeutic implications of slightly elevated serum bilirubin concentrations, we will discuss the recent prospective studies on cardiovascular risk in individuals with Gilbert syndrome (GS) as well as those with the UGT1A1*28 allele. Such individuals have decreased hepatic bilirubin UDP-glucuronosyltransferase activity, decreased bilirubin clearance, and increased serum bilirubin concentrations. Lastly, we will discuss some of the therapeutic approaches that could be used to increase serum bilirubin concentrations to prevent CVD and other oxidative and inflammatory diseases.

High-performance liquid chromatographic analysis of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol in ginger-containing dietary supplements, spices, teas, and beverages.

Ginger root powder is widely used as a dietary supplement as well as a spice and flavoring agent in foods and beverages. In this study, we developed a high-performance liquid chromatographic (HPLC) method that is suitable for the analysis of 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol in a wide variety of ginger-containing dietary supplements, spices, teas, mints, and beverages. 6-Gingerol, 6-shogaol, 8-gingerol, and 10-gingerol were extracted from various ginger-containing products with ethyl acetate and analyzed by HPLC on a C-8 reversed phase column at 282 nm. The recoveries of 6-, 8-, and 10-gingerol, and 6-shogaol from the ginger dietary supplements and ginger-containing products were 94.7+/-4.1, 93.6+/-3.4, 94.9+/-4.0, 97.1+/-3.8%, respectively. The within-day coefficients of variation for 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol standards at 50.0 microg/mL were 2.54, 2.38, 2.55, and 2.31%, respectively. The lower limit of quantitation was 25 ng injected. The standard curves for 6-, 8-, and 10-gingerol and 6-shogaol were linear from 10.0 to 1000 microg/mL. The variation (CV's) in the 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol concentrations of nine different ginger root dietary supplements were 115.2, 45.7, 72.3, and 141.7%, respectively. The gingerol composition of various ginger-containing spices, teas, and beverages also were found to vary widely. The proposed method can be used for the analysis and standardization of 6-, 8-, and 10-gingerol in ginger-containing dietary supplements, spices, food products and beverages and as a method for determining the amounts of 6-shogaol as a marker for 6-gingerol stability.

Comparison of urine and serum testing for early detection of acetaminophen ingestion.

Acetaminophen is a common ingestant that is routinely screened for, with serum testing, after overdoses. We compared a modified, qualitative, colorimetric, o-cresol urine test for acetaminophen with the standard laboratory serum immunoassay. Twenty-nine adult volunteers were given 975 mg of acetaminophen, and then serum and urine samples were tested at predefined intervals for up to 4 hours. The presence of acetaminophen in the urine samples was determined with a modified o-cresol assay by two independent reviewers and was verified by using high-performance liquid chromatography. Compared with the acetaminophen serum immunoassay, the acetaminophen urine test had specificity of 97% (95% confidence interval, 80-100%) and sensitivity of 100% (95% confidence interval, 81-100%) at 45 minutes. Further studies to improve and to accelerate processing of the qualitative colorimetric o-cresol urine test could allow the test to be used in specific military and civilian clinical scenarios.

The heme catabolic pathway and its protective effects on oxidative stress-mediated diseases.

Bilirubin, the principal bile pigment, is the end product of heme catabolism. For many years, bilirubin was thought to have no physiological function other than that of a waste product of heme catabolism--useless at best and toxic at worst. Although hyperbilirubinemia in neonates has been shown to be neurotoxic, studies performed during the past decade have found that bilirubin has a number of new and interesting biochemical and biological properties. In addition, there is now a strong body of evidence suggesting that bilirubin may have a beneficial role in preventing oxidative changes in a number of diseases including atherosclerosis and cancer, as well as a number of inflammatory, autoimmune, and degenerative diseases. The results also suggest that activation of the heme oxygenase and heme catabolic pathway may have beneficiary effects on disease prevention either through the action of bilirubin or in conjunction with bilirubin. If so, it may be possible to therapeutically induce heme oxygenase, increase bilirubin concentrations, and lower the risk of oxidative stress-related diseases.

Variation in concentration and labeling of ginger root dietary supplements.

OBJECTIVE: Ginger root dietary supplements are often used to alleviate symptoms of nausea and vomiting associated with pregnancy. In this study, we determined the variation in 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol concentrations and labeling of different brands of ginger root dietary supplements. METHODS: Ten different ginger root dietary supplements were purchased randomly at local pharmacies and health food stores. The 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol concentrations of the dietary supplements were determined by high-performance liquid-chromatography. In addition, we examined the container labeling for the amount of ginger root powder or extract in each capsule, the serving size, ingredients, expiration date, lot number, standardization procedure, and suggested use. RESULTS: The 6-gingerol concentration of the ginger powder dietary supplements ranged from 0.0 to 9.43 mg/g, (mean +/- standard deviation, 2.56 +/- 2.95 mg/g), 6-shogaol ranged from 0.16 to 2.18 mg/g (1.27 +/- 0.58), 8-gingerol ranged from 0.00 to 1.1 mg/g (0.47 +/- 0.34), and 10-gingerol ranged from 0.00 to 1.40 mg/g (0.36 +/- 0.51). The amounts of 6-gingerol, 6-shogaol, 8-gingerol, and 10-gingerol in the ginger root dietary supplements varied widely on both a milligram per gram basis and on a milligram per capsule basis. Likewise, the suggested ginger serving sizes varied from 250 mg to 4.77 g per day. CONCLUSION: The results of this study indicate that there is a wide variation in the gingerol composition and in the suggested serving sizes of ginger root powder from different manufacturers. LEVEL OF EVIDENCE: II-3.

Comparison of actual and stated concentrations of pharmaceuticals manufactured in Mexico.

BACKGROUND: During the past 5 years, there has been much concern about the safety of and ease of access to drugs that are unavailable without a prescription in the United States but are manufactured and can be purchased over the counter (OTC) in Mexico. However, based on a literature search, studies have not been performed to determine whether such drugs meet the US Pharmacopoeia (USP) standards for percent of labeled amount. OBJECTIVE: The aim of this study was to determine whether 5 drugs commonly prescribed in the United States and manufactured and purchased in Mexico met USP standards for stated concentration, were properly packaged and sealed, and had properly labeled expiration dates and lot numbers. METHODS: For the study, we purchased acetaminophen, carbamazepine, diazepam, digoxin, and phenytoin from 2 to 4 pharmacies in Ciudad Acuna, Mexico. Samples of each drug, each with a different lot number, were purchased OTC or with a prescription. None of the pharmacists had knowledge of the study. Brand-name or generic drugs manufactured and obtained in the United States were also analyzed for comparative purposes. Each drug was analyzed by high-performance liquid chromatography using USP guidelines established for the analysis of each drug. In addition, we visually examined the packaging, seals, color, and appearance of the drugs, and observed whether the packages contained lot numbers and whether the drugs were within the stated expiration dates. RESULTS: For all 5 drugs, the 95% CIs fell within the USP-defined acceptable ranges for percent of labeled amount. This was found to be true for drugs obtained at different pharmacies, for different drug manufacturers, and for drugs with different lot numbers. All of the drugs were found to be properly packaged and sealed, had lot numbers listed on the packages, and were sold before the stated expiration dates. CONCLUSION: Although health care practitioners should be aware that patients might be using drugs manufactured and purchased in Mexico, the results of this study of a small sample of such drugs suggest they meet the USP standards for percent of labeled amount.

Determination of modafinil in plasma and urine by reversed phase high-performance liquid-chromatography.

Modafinil (Provigil) is a new wake-promoting drug that is being used for the management of excessive sleepiness in patients with narcolepsy. It has pharmacological properties similar to that of amphetamine, but without some of the side effects associated with amphetamine-like stimulants. Since modafinil has the potential to be abused, accurate drug-screening methods are needed for its analysis. In this study, we developed a high-performance liquid-chromatographic procedure (HPLC) for the quantitative analysis of modafinil in plasma and urine. (Phenylthio)acetic acid was used as an internal standard for the analysis of both plasma and urine. Modafinil was extracted from urine and plasma with ethyl acetate and ethyl acetate-acetic acid (100:1, v/v), respectively, and analyzed on a C18 reverse phase column with methanol-water-acetic acid (500:500:1, v/v) as the mobile phase. Recoveries from urine and plasma were 80.0 and 98.9%, respectively and the limit of quantitation was 0.1 microg/mL at 233 nm. Forty-eight 2-h post-dose urine samples from sham controls and from individuals taking 200 or 400 mg of modafinil were analyzed without knowledge of drug administration. All 16-placebo urine samples and all 32 2-h post-dose urine samples were correctly classified. The analytical procedure is accurate and reproducible and can be used for therapeutic drug monitoring, pharmacokinetic studies, and drug abuse screening.