Additional value of advanced neurosonography and magnetic resonance imaging in fetuses at risk for brain damage.

OBJECTIVE: To assess the additional value of fetal multiplanar (axial, coronal and sagittal) neurosonography and magnetic resonance imaging (MRI) to that of the standard axial ultrasound planes in diagnosing brain damage in fetuses at high risk. METHODS: This was a prospective, multicenter, observational study. Women were eligible for participation if their fetus was at risk for acquired brain anomalies. Risk factors were congenital infection, alloimmune thrombocytopenia, fetal growth restriction, trauma during pregnancy, fetal hydrops, monochorionic twins and prior ultrasound finding suggestive of an acquired brain anomaly. Examinations of the fetal brain before birth comprised axial ultrasound and advanced neurosonography biweekly and MRI once. After birth, neonatal cranial ultrasound was performed at < 24 h and at term-equivalent age. Neonatal brain MRI was performed once at term-equivalent age. An expert panel blinded to medical information, including imaging findings by the other methods, evaluated the presence of periventricular echogenicity (PVE) changes, peri- and intraventricular hemorrhage (IVH) and changes in basal ganglia and/or thalami echogenicity (BGTE) on ultrasound, and the equivalent signal intensity (SI) changes on MRI. Conclusions on imaging findings were generated by consensus. The children were followed up with examinations for psychomotor development at 1 year of age, using the Touwen examination and Alberta Infant Motor Scale, and at 2 years of age using Bayley Scale of Infant Development-III (BSID-III) and behavioral, sensory profile and linguistic questionnaires; scores > 1 SD below the mean were considered suspicious for neurodevelopmental sequelae. RESULTS: Fifty-six fetuses were examined, and in 39/56 fetuses, all fetal-imaging modalities were available. PVE/SI changes were observed in 6/39, 21/39 and 2/39 fetuses on axial ultrasound planes, multiplanar neurosonography and MRI, respectively. IVH was found in 3/39, 11/39 and 1/39 fetuses, and BGTE/SI changes in 0/39, 12/39 and 0/39 fetuses, respectively. Outcome was suspicious for neurodevelopmental sequelae in 13/46 infants at 1 year, and at 2 years, 41/41 children had scores within 1 SD of the mean on BSID-III and 20 had scores > 1 SD below the mean on the behavioral (5/38), sensory profile (17/37) and/or linguistic (6/39) questionnaires. CONCLUSIONS: In this cohort of fetuses at risk for brain damage, the severity of acquired brain anomalies was limited. Nevertheless, multiplanar neurosonography detected more fetal PVE changes, IVH and/or BGTE changes compared to the standard axial ultrasound planes and MRI. Fetal MRI did not demonstrate any anomalies that were not seen on neurosonography. Neurodevelopmental outcome at 2 years of age showed no or mild impairment in most cases. © 2019 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Mood configurations and their relationship to immune system responses: Exploring the relationship between moods, immune system responses, thyroid hormones, and social support.

Analyzing data on 2,057 healthy subjects in the Dutch Lifelines database we explore the relationship between immune system responses, thyroid hormone functioning and people's mood that is expected to be moderated by social support. We focus (1) on the innate immune system cell count: monocytes, eosinophil granulocytes, basophilic granulocytes, neutrophil granulocytes; and thrombocytes; and (2) on the adaptive immune system cell count: lymphocytes (T, B and NK cells). Moods were measured on the positive (PA) and negative (NA) dimensions of the PANAS scale, divided in four groups based on their PA and NA median scores: hedonic, positive mood, negative mood and anhedonic. We focus further on (3) thyroid cells: T3 and T4; and (4) on social support. We found significant differences between mood groups and mean cell counts for basophilic granulocytes and thrombocytes but not for monocytes, eosinophil granulocytes and neutrophil granulocytes in the innate immune system. However, in the adaptive immune system we found mean lymphocyte cell counts to be different in all four mood groups. We also found that T3 and T4 levels differ significantly across all mood groups and work in very close association with lymphocytes to activate the adaptive immune system. These differences were most significant in the hedonic and anhedonic groups. The findings allow us to better understand mood groups, especially the hedonic and anhedonic groups, and open up new avenues for intervention.

Diagnostic accuracy of preoperative ultrasonography in predicting contralateral inguinal hernia in children: a systematic review and meta-analysis.

OBJECTIVES: The incidence of children developing metachronous contralateral inguinal hernia (MCIH) is 7-15%. Contralateral groin exploration during unilateral hernia repair can prevent MCIH development and subsequent second surgery and anaesthesia. Preoperative ultrasonography is a less invasive strategy and potentially able to detect contralateral patent processus vaginalis (CPPV) prior to MCIH development. METHODS: We queried MEDLINE, Embase and Cochrane library to identify studies regarding children aged < 18 years diagnosed with unilateral inguinal hernia without clinical signs of contralateral hernia, who underwent preoperative ultrasonography of the contralateral groin. We assessed heterogeneity and used a random-effects model to obtain pooled estimates of sensitivity, specificity and area under the receiver operating characteristic curve (AUC). RESULTS: Fourteen studies (2120 patients) were included, seven (1013 patients) in the meta-analysis. In studies using surgical exploration as reference test (n = 4, 494 patients), pooled sensitivity and specificity were 93% and 88% respectively. In studies using contralateral exploration as reference test following positive and clinical follow-up after negative ultrasonographic test results (n = 3, 519 patients), pooled sensitivity was 86% and specificity 98%. The AUC (0.984) shows high diagnostic accuracy of preoperative ultrasonography for detecting CPPV, although diagnostic ultrasonographic criteria largely differ and large heterogeneity exists. Reported inguinal canal diameters in children with CPPV were 2.70 ± 1.17 mm, 6.8 ± 1.3 mm and 9.0 ± 1.9 mm. CONCLUSION: Diagnostic accuracy of preoperative ultrasonography to detect CPPV seems promising, though may result in an overestimation of MCIH prevalence, since CPPV does not invariably lead to MCIH. Unequivocal ultrasonographic criteria are mandatory for proper diagnosis of CPPV and subsequent prediction of MCIH. KEY POINTS: • Diagnostic accuracy of preoperative ultrasonography for detection of CPPV in children with unilateral inguinal hernia is high. • Preoperative ultrasonographic evaluation of the contralateral groin assumedly results in an overestimation of MCIH prevalence. • Unequivocal ultrasonographic criteria are mandatory for proper diagnosis of CPPV and risk factor identification is needed to predict whether CPPV develops into clinically apparent MCIH.

Infection dynamics across the dry period using Dairy Herd Improvement somatic cell count data and its effect on cow performance in the subsequent lactation.

In this study, we studied infection dynamics across the dry period using test-day somatic cell count (SCC) data from 739 Holstein cows from 33 randomly selected commercial dairy herds in Flanders, all of which applied blanket dry-cow therapy at dry-off. First, we determined infection dynamics, combining the last test-day SCC before dry-off and the first test-day SCC after calving. Next, we determined the effect of dry period infection dynamics, adjusting for the level of the second test-day SCC after calving, on the evolution of test-day SCC and milk yield (MY) and on clinical mastitis and culling hazard in the subsequent lactation. Using an SCC threshold of 200,000 cells/mL, 12.6% of the cows considered healthy before dry-off acquired a new intramammary infection (IMI) across the dry period, whereas 66.9% of the cows considered infected before dry-off cured from IMI. Infection dynamics across the dry period significantly affect a cow's SCC, clinical mastitis risk, and culling hazard in the subsequent lactation. Cows with a new IMI, a cured IMI, or a chronic IMI across the dry period had higher test-day SCC than healthy cows, and their test-day SCC evolved differently over time. This was not the case for test-day milk yield, for which no association with infection dynamics was detected. Furthermore, cows with a second test-day SCC <200,000 cells/mL had a lower test-day SCC in the remainder of the lactation than cows with a second test-day SCC ≥200,000 cells/mL, but this association was modified by infection dynamics across the dry period. The lowest test-day SCC in the remainder of the lactation was observed for cows that remained healthy across the dry period combined with a low (<200,000 cells/mL) second test-day SCC. Cows that cured from an IMI present at dry-off and cows with a chronic IMI across the dry period were more likely to develop clinical mastitis (hazard ratio = 2.22 and 2.89; 95% confidence interval = 1.45-3.43 and 1.60-5.20, respectively), and chronic IMI cows were more likely to be culled (hazard ratio = 3.68; 95% confidence interval = 1.64-8.20) in the subsequent lactation compared with healthy cows. This was not true for cows that became infected across the dry period. This study underlines the importance of good udder health management during lactation to prevent IMI at dry-off rather than curing infected cows during the dry period to ensure optimal udder health in the subsequent lactation.

Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine.

An experimental trial was conducted to explore the effect of vaccination with a polyvalent vaccine against mastitis (Startvac) on the early immune response after experimental intramammary challenge with a heterologous killed Staphylococcus aureus strain. The effect of vaccination on milk production, clinical signs, quarter milk somatic cell count, milk polymorphonuclear neutrophilic leukocyte (PMN) concentration and viability, the concentration of antigen-specific antibodies [slime associated antigenic complex (SAAC) and J5] and their IgG1 and IgG2 subtypes in both serum and whey, and the antigen-specific IFN-γ, IL-4, and IL-17 production by blood lymphocytes after in vitro stimulation with S. aureus and Escherichia coli extracts were determined. A cohort of 8 clinically healthy end-term cows and heifers were conveniently selected, of which half was vaccinated with Startvac at 45 and 10 d before the expected calving date and half served as nonvaccinated control animals. At 15 d in milk, 2 contralateral quarters of each of the 8 animals were challenged with 2×109 cfu/mL of the formaldehyde-killed S. aureusC195strain. The 2 other quarters were infused with phosphate-buffered saline and served as control quarters. The increase in both quarter milk somatic cell count and PMN concentration and the drop in milk production after S. aureus inoculation was less pronounced in the vaccinates than in the nonvaccinates, reflecting a less severe inflammatory response. No significant differences in PMN viability between vaccinates and nonvaccinates could be demonstrated. The serum SAAC- and J5-specific antibody concentration significantly increased across the dry period in the vaccinated animals only. The whey concentration of SAAC-specific antibodies was significantly higher in vaccinates than in nonvaccinates at both 15 and 17 d in milk, independent from the challenge status of the quarters. No significant differences in the whey J5-specific antibody concentration were observed. Vaccination with Startvac seems to primarily evoke a Th2 response for S. aureus characterized by a shift toward the IgG1 antibody subtype and accompanied by a less pronounced Th1 response. The type of response against E. coli was less clear, though a weak but significant shift toward the IgG2 antibody subtype after vaccination and high IFN-γ levels after in vitro stimulation suggest a Th1 response. The increased SAAC-specific antibody concentration in whey in vaccinates compared with nonvaccinates most probably triggers the opsonization of the inoculated S. aureus bacteria, resulting in a more efficient elimination of the bacteria from the mammary gland.

Milk production, quality, and consumption in Jimma (Ethiopia): Facts and producers', retailers', and consumers' perspectives.

Four studies were performed to quantify milk production, quality and consumption in the town Jimma, Ethiopia. First, 47 dairy farmers and 44 milk retailers were interviewed to gain more insights in dairy farming and marketing, and associated constraints. Second, bulk milk samples (n=188) were collected for 4 consecutive weeks to investigate milk quality [Total Bacterial Counts (TBC), Coliform Counts (CC), Somatic Cell Counts (SCC), and antimicrobial residues]. Third, (bulk) milk samples from 32 farms, 46 milk retailers and the 3 local milk collection centers were collected to determine the presence of oxacillin susceptible-and oxacillin resistant Staphylococcus aureus. Fourth, 208 adult inhabitants were interviewed to gain more insight in milk consumption and associated concerns of consumers. The average dairy farm included in the studies consisted of 5 lactating cows, produced 43 liters of milk per day and was owned by male, literate adults. Milk was sold to retailers (71% of the production) and directly to customers (25%) without any quality control, whereas 4% was self-consumed. Shortage of animal nutrition and adulteration of the milk were the main constraints for farmers and retailers, respectively. The median TBC, CC and SCC were 122,500CFU/mL, 1,005CFU/mL and 609,500cells/mL, respectively. Antimicrobial residues were detected in 20% of all samples. In general, the milk quality was considered to be poor (TBC>10,000CFU/mL, and/or CC>100CFU/mL, and/or SCC>400,000cells/mL and/or presence of antimicrobial residues) in 97% of all samples. S. aureus was isolated from 12 (38%), 13 (33%), and 2 out of 3 of the milk samples originating from the dairy farms, the milk retailers, and the milk collection centers, respectively. Seven (26%) of the isolates were resistant to oxacillin suggesting the presence of MRSA (Lee, 2003). Local milk is occasionally consumed by adults but more frequently by children. Adults mainly drink spontaneously fermented milk (57% of 105 interviewees consuming local milk) whereas most milk for children is boiled (86% of 110 households with children consuming local milk). Most consumers are concerned about adulteration and milk borne diseases but not about antimicrobial residues. Educated consumers (secondary school or higher) were more likely to boil milk for own consumption, to be concerned about antimicrobial residues in milk, to be concerned about milk borne diseases and to be willing to pay more for milk with proven good quality compared to poorly educated consumers. We conclude that milk quality incentives should be introduced in Jimma, and investments should be made in knowledge transfer, training, milk collection systems and a central milk quality lab.

Pathogen group specific risk factors for clinical mastitis, intramammary infection and blind quarters at the herd, cow and quarter level in smallholder dairy farms in Jimma, Ethiopia.

A cross-sectional study on clinical mastitis, intramammary infection (IMI) and blind quarters was conducted on 50 smallholder dairy farms in Jimma, Ethiopia. A questionnaire was performed, and quarters of 211 cows were sampled and bacteriologically cultured. Risk factors at the herd, cow, and quarter level for clinical mastitis and (pathogen-specific) intramammary infection were studied using multilevel modeling. As well, factors associated with quarters being blind were studied. Eleven percent of the cows and 4% of the quarters had clinical mastitis whereas 85% of the cows and 51% of the quarters were infected. Eighteen percent of the cows had one or more blind quarter(s), whereas 6% of the quarters was blind. Non-aureus staphylococci were the most frequently isolated pathogens in both clinical mastitis cases and IMI. The odds of clinical mastitis was lower in herds where heifers were purchased in the last year [odds ratio (OR) with 95% confidence interval: 0.11 (0.01-0.90)], old cows (>4 years) [OR: 0.45 (0.18-1.14)], and quarters not showing teat injury [OR: 0.23 (0.07-0.77)]. The odds of IMI caused by any pathogen was higher in herds not practicing teat drying before milking (opposed to drying teats with 1 towel per cow) [OR: 1.68 (1.05-2.69)], cows in later lactation (>180 DIM opposed to ≤90 DIM) [OR: 1.81 (1.14-2.88)], cows with a high (>3) body condition score (BCS) [OR: 1.57 (1.06-2.31)], right quarters (opposed to a left quarter position) [OR: 1.47 (1.10-1.98)], and quarters showing teat injury [OR: 2.30 (0.97-5.43)]. Quarters of cows in herds practicing bucket-fed calf feeding (opposed to suckling) had higher odds of IMI caused by Staphylococcus aureus [OR: 6.05 (1.31-27.90)]. Except for BCS, IMI caused by non-aureus staphylococci was associated with the same risk factors as IMI caused by any pathogen. No access to feed and water immediately after milking [OR: 2.41 (1.26-4.60)], higher parity [OR: 3.60 (1.20-10.82)] and tick infestation [OR: 2.42 (1.02-5.71)] were risk factors for quarters being blind. In conclusion, replacement of old cows, prevention of teat injuries/lesions, drying teats with 1 towel per cow before milking, improving fertility in order to shorten the lactation period, allowing (restricted) suckling, access to feed and water immediately after milking, and improving tick control could improve udder health in Jimma.

Milk prolactin response and quarter milk yield after experimental infection with coagulase-negative staphylococci in dairy heifers.

Coagulase-negative staphylococci (CNS) are the most common bacteria involved in subclinical mastitis in dairy cows. Remarkably, CNS-infected dairy heifers produce more milk than uninfected heifers. Because the lactation hormone prolactin (PRL) is also involved in mammary gland immunity, we investigated the milk PRL response and the mammary quarter milk yield following experimental CNS challenge. Eight healthy Holstein-Friesian heifers in mid-lactation were experimentally infected using a split-udder design with 3 different CNS strains: one Staphylococcus fleurettii (from sawdust bedding) and 2 Staphylococcus chromogenes strains (one isolate from a teat apex, the other isolate from a chronic intramammary infection). Three mammary quarters per heifer were simultaneously inoculated with 1.0×10(6) cfu, whereas the remaining mammary quarter was infused with sterile phosphate-buffered saline, serving as a control. An existing radioimmunoassay was modified, validated, and used to measure PRL frozen-thawed milk at various time points until 78h after challenge. The mean milk PRL level tended to be higher in the CNS-challenged mammary quarters compared with the control mammary quarters (7.56 and 6.85ng/mL, respectively). The increase in PRL over time was significantly greater in the CNS-challenged mammary quarters than in the control mammary quarters. However, no difference was found in the PRL response when comparing each individual CNS strain with the control mammary quarters. The mean mammary quarter milk yield tended to be lower in the CNS-infected mammary quarters than in the control mammary quarters (1.73 and 1.98kg per milking, respectively). The greatest milk loss occurred in the mammary quarters challenged with the intramammary strain of S. chromogenes. Future observational studies are needed to elucidate the relation between PRL, the milk yield, and the inflammatory condition, or infection status, of the mammary gland.

Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica.

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform ß-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ∆12 desaturase (Fad2p). We also expressed the Ricinus communis ∆12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7% of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29% of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35% of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14% of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43% of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.

Risk factors associated with subclinical mastitis as detected by California Mastitis Test in smallholder dairy farms in Jimma, Ethiopia using multilevel modelling.

The prevalence of subclinical mastitis and associated risk factors at the herd, cow and quarter level were studied using multilevel modelling in smallholder dairy farms in Jimma, Ethiopia. Forty-two herds, out of the 55 dairy farms located in Jimma (76%), were visited, a questionnaire was performed, and 635 quarters belonging to 176 lactating cows were screened to detect the presence of subclinical mastitis using the California Mastitis Test (CMT). Sixty-two percent of the cows and 51% of the quarters were subclinically infected. Quarters from cows in later stage of lactation (>180 DIM) [opposed to early lactation (<90 DIM)] were more likely to be subclinically infected (OR=2.40, 95% CI=1.44-3.99). Overall, quarters from cows milked by squeezing (as opposed to stripping) were less likely to be subclinically infected (OR=0.45, 95% CI=0.29-0.71), although quarters from cows with tick-infested udders were more likely to be subclinically infected when milked by squeezing (as opposed to stripping). The milking technique did not influence the likelihood of infection in cows without ticks on the udder. This study stresses the high prevalence of subclinical mastitis in smallholder dairy farms in Jimma and a lack of awareness of the existence of the disease among dairy farmers. Implementation of a mastitis prevention program adapted to the local needs, including a focus on milking technique, application of appropriate tick control measurements as well as fertility management, allowing cows to be dried-off at a more appropriate moment, are needed. To conclude, milking by squeezing instead of stripping, but not of cows with tick-infested udders, as well as fertility management could reduce the subclinical mastitis prevalence (and incidence) on the short term.

Accuracy profiles assessing the validity for routine use of high-performance thin-layer chromatographic assays for drug formulations.

The accuracy profile, based on total error, integrates several validation parameters, such as trueness, precision and linearity, providing one statistic which enables decision on the suitability of a method for its intended purpose. Two assay methods for formulations are validated using accuracy profiles as an alternative approach to classic method validation. It concerns high-performance thin-layer chromatography (HPTLC) methods, which initially were validated using the classic approach. The first method assayed sulfamethoxazole and trimethoprim, and the second lamivudine, stavudine and nevirapine. Both formulations are fixed-dose combination tablets. The resulting accuracy profiles showed that the 95% ß-expectation tolerance limits for all compounds fell well within the bias acceptance limits set at ±5%. This means that the two analytical thin-layer chromatographic methods are capable of making accurate results at the studied concentration ranges of each compound. Measurement uncertainties of every compound at each concentration level could also be determined from the accuracy profile data.

Optimization of a reversed-phase-high-performance thin-layer chromatography method for the separation of isoniazid, ethambutol, rifampicin and pyrazinamide in fixed-dose combination antituberculosis tablets.

This paper presents the development of a new RP-HPTLC method for the separation of pyrazinamide, isoniazid, rifampicin and ethambutol in a four fixed-dose combination (4 FDC) tablet formulation. It is a single method with two steps in which after plate development pyrazinamide, isoniazid and rifampicin are detected at an UV wavelength of 280 nm. Then ethambutol is derivatized and detected at a VIS wavelength of 450 nm. Methanol, ethanol and propan-1-ol were evaluated modifiers to form alcohol-water mobile phases. Systematic optimization of the composition of each alcohol in the mobile phase was carried out using the window diagramming concept to obtain the best separation. Examination of the Rf distribution of the separated compounds showed that separation of the compounds with the mobile phase containing ethanol at the optimal fraction was almost situated within the optimal Rf-values region of 0.20-0.80. Therefore, ethanol was selected as organic modifier and the optimal mobile phase composition was found to be ethanol, water, glacial acetic acid (>99% acetic acid) and 37% ammonia solution (70/30/5/1, v/v/v/v). The method is new, quick and cheap compared to the actual method in the International Pharmacopoeia for the assay of the 4 FDC tablets, which involves the use of two separate HPLC methods.

Similarity analyses of chromatographic fingerprints as tools for identification and quality control of green tea.

Similarity assessment of complex chromatographic profiles of herbal medicinal products is important as a potential tool for their identification. Mathematical similarity parameters have the advantage to be more reliable than visual similarity evaluations of often subtle differences between the fingerprint profiles. In this paper, different similarity analysis (SA) parameters are applied on green-tea chromatographic fingerprint profiles in order to test their ability to identify (dis)similar tea samples. These parameters are either based on correlation or distance measurements. They are visualised in colour maps and evaluation plots. Correlation (r) and congruence (c) coefficients are shown to provide the same information about the similarity of samples. The standardised Euclidean distance (ds) reveals less information than the Euclidean distance (de), while Mahalanobis distances (dm) are unsuitable for the similarity assessment of chromatographic fingerprints. The adapted similarity score (ss*) combines the advantages of r (or c) and de. Similarity analysis based on correlation is useful if concentration differences between samples are not important, whereas SA based on distances also detects concentration differences well. The evaluation plots including statistical confidence limits for the plotted parameter are found suitable for the evaluation of new suspected samples during quality assurance. The ss* colour maps and evaluation plots are found to be the best tools (in comparison to the other studied parameters) for the distinction between deviating and genuine fingerprints. For all studied data sets it is confirmed that adequate data pre-treatment, such as aligning the chromatograms, prior to the similarity assessment, is essential. Furthermore, green-tea samples chromatographed on two dissimilar High-Performance Liquid Chromatography (HPLC) columns provided the same similarity assessment. Combining these complementary fingerprints did not improve the similarity analysis of the studied data set.

HPTLC methods to assay active ingredients in pharmaceutical formulations: a review of the method development and validation steps.

High-performance thin-layer chromatography (HPTLC) is still increasingly finding its way in pharmaceutical analysis in some parts of the world. With the advancements in the stationary phases and the introduction of densitometers as detection equipment, the technique achieves for given applications a precision and trueness comparable to high-performance liquid chromatography (HPLC). In this review, the literature is surveyed for developed and validated HPTLC methods to assay active ingredients in pharmaceutical formulations published in the period 2005-2011. Procedures and approaches for method development, validation and quantitative assays are compared with the standard ways of conducting them. Applications of HPTLC in some other areas are also briefly highlighted.

Development and validation of a normal-phase HPTLC method for the simultaneous analysis of lamivudine, stavudine and nevirapine in fixed-dose combination tablets.

This paper presents the development and validation of an improved method for the simultaneous analysis of lamivudine (LVD), stavudine (STV) and nevirapine (NVP) using high-performance thin-layer chromatography (HPTLC) with densitometric detection. Separation was performed on silica gel 60F(254) plates. The mobile phase is comprised of ethylacetate, methanol, toluene and concentrated ammonia (38.7:19.4:38.7:3.2, v:v:v:v). Detection wavelength was 254 nm. The R(f) values were 0.24±0.03, 0.38±0.04 and 0.69±0.04 (n=8) for LVD, STV and NVP, respectively. An F-test indicated that calibration graphs were adequately linear at the evaluated concentration ranges. The pooled %RSD for repeatability of the percentage amount recovered for LVD, STV and NVP were found to be 0.62, 0.54, and 0.79, and the pooled %RSD for time-different intermediate precision were 1.66, 1.27 and 1.21. The percentage recoveries for the trueness were 99.2%±1.5 for LVD, 98.6%±1.5 for STV and 99.3%±1.7 for NVP (n=3). Most factors evaluated in the robustness test were found to have an insignificant effect on the selected responses at 95% confidence level. This method was successfully used to analyze fixed-dose tablets samples of LVD, STV and NVP.

Exploratory analysis of chromatographic fingerprints to distinguish rhizoma Chuanxiong and rhizoma Ligustici.

Identification and quality control of products of natural origin, used for preventive and therapeutical goals, is required by regulating authorities, as the World Health Organization. This study focuses on the identification and distinction of the rhizomes from two Chinese herbs, rhizoma Chuanxiong (from Ligusticum chuanxiong Hort.) and rhizoma Ligustici (from Ligusticum jeholense Nakai et Kitag), by chromatographic fingerprints. A second goal is using the fingerprints to assay ferulic acid, as its concentration provides an additional differentiation feature. Several extraction methods were tested, to obtain the highest number of peaks in the fingerprints. The best results were found using 76:19:5 (v/v/v) methanol/water/formic acid as solvent and extracting the pulverized material on a shaking bath for 15 min. Then fingerprint optimization was done. Most information about the herbs, i.e. the highest number of peaks, was observed on a Hypersil ODS column (250 mm × 4.6 mm ID, 5 µm), 1.0% acetic acid in the mobile phase and employing within 50 min linear gradient elution from 5:95 (v/v) to 95:5 (v/v) acetonitrile/water. The final fingerprints were able to distinguish rhizoma Chuanxiong and Ligustici, based on correlation coefficients combined with exploratory data analysis. The distinction was visualized using Principal Component Analysis, Projection Pursuit and Hierarchical Clustering Analysis techniques. Quantification of ferulic acid was possible in the fingerprints of both rhizomes. The time-different intermediate precisions of the fingerprints and of the ferulic acid quantification were shown to be acceptable.

Brain abnormalities on MR imaging in patients with retinoblastoma.

BACKGROUND AND PURPOSE: Although pineoblastoma is the main brain abnormality associated with hereditary retinoblastoma, recent studies suggest an association with pineal cysts. This association is important because some pineoblastomas mimic pineal cysts. If there is a relationship, then radiologists should be aware of it because diagnostic confusion is possible. Mental retardation and congenital brain anomalies are also reported in patients with retinoblastoma, mostly in combination with 13q deletion syndrome. In this retrospective study, the presence of brain abnormalities on MR images in a large group of consecutive patients with retinoblastoma is evaluated. MATERIALS AND METHODS: Brain MR images of 168 patients with retinoblastoma from 1989 to 2009 were evaluated by 2 radiologists for tumors, structural anomalies, myelinization, and coincidental findings. Clinical records were reviewed for laterality, heredity, and the presence of the 13q deletion syndrome. RESULTS: The hereditary group (patients with bilateral and unilateral proved RB1-germline mutation) included 90 (54%) of 168 patients. Seven patients had 13q deletion syndrome. Normal findings on brain MR images were seen in 150 (89%) patients. Five pineoblastomas were detected, all in patients with hereditary retinoblastoma (5.5% in the hereditary subgroup). Nine pineal cysts were detected (2.2% in the hereditary subgroup). Corpus callosum agenesis was found in 1 patient and a Dandy-Walker variant in 1 patient, both in combination with 13q deletion syndrome. CONCLUSIONS: Pineoblastoma is associated with hereditary retinoblastoma, and structural brain abnormalities are restricted to patients with the 13q deletion syndrome. The incidence of pineal cysts in patients with retinoblastomas is similar to that in healthy children and is not associated with hereditary retinoblastoma.

Recognizing paracetamol formulations with the same synthesis pathway based on their trace-enriched chromatographic impurity profiles.

The development of a new drug substance is an expensive and time-consuming process. Therefore, the developers want to maximize the profit from the drug by patenting the concerned molecule as well as its synthesis pathway. In a later stage a faster or cheaper manufacturing process can be developed and patented. The aim of this study is to recognize paracetamol-containing drug formulations in relation to their synthesis pathways, in order to demonstrate the possibility to reveal fraudulently synthesized paracetamol. Since different synthesis pathways require different starting materials, solvents, reagents and catalysts and since they can produce different intermediates, it is expected that drug products originating from a different synthesis pathway will exhibit a different impurity profile. Therefore, in this study several paracetamol samples, synthesized in four different ways, are analyzed using trace-enrichment high-performance liquid chromatography (HPLC). The resulting chromatographic data were chemometrically treated in order to reveal clustering tendencies in the hope of distinguishing the different pathways. When performing principal component analysis (PCA) only 3 vaguely outlined clusters appeared. Projection pursuit (PP) was able to reveal 4 clusters and the samples with known synthesis pathway, except one, were classified in the different clusters. When hierarchical clustering and auto-associative multivariate regression trees (AAMRT) were applied, the samples of the four synthesis pathways could also be distinguished. AAMRT has an added value, since it can indicate the variables (peaks and thus also the impurities) that are responsible for the differences between the samples synthesized differently.

Development and validation of a normal-phase high-performance thin layer chromatographic method for the analysis of sulfamethoxazole and trimethoprim in co-trimoxazole tablets.

Pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals, such as HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of sulfamethoxazole (SMX) and trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets within resource-limited countries, Medicines Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets. SMX and TMP were separated on glass-backed silica gel 60 F(254) plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase was comprised of toluene, ethylacetate and methanol (50:28.5:21.5, v:v:v). Detection wavelength was 254 nm. The R(f) values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. Cochran's criterion test indicated homoscedasticity of variances for the calibration data. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time-different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00%+/-1.83 and 99.66%+/-1.21 for SMX and TMP, respectively. The method was found to be robust and was then successfully applied to analyze co-trimoxazole tablet samples.