Effectiveness of silver diamine fluoride in arresting early childhood caries: a randomised controlled clinical trial.

AIM: Evaluate the effectiveness of biannual 38% SDF in arresting early childhood caries (ECC) compared to atraumatic restorative treatment (ART). METHODS: One hundred children (aged 2-5 years) with ECC were (2-5 years) divided equally and randomly into two groups. Test group: 38% SDF biannually; Control group: ART. The primary outcome was caries arrestarrest, which was assessed at the 3,6,9, and 12 months interval. Oral health- related quality of life (OHRQoL) was evaluated at base line and after 12 months using the Parental-Caregiver Perceptions Questionnaire (P-CPQ) and Family Impact Scale (FIS). CONCLUSION: SDF was considerably more effective in arresting ECC after 12 months compared to ART. An overall significant improvement of OHRQoL was reported for both groups with SDF being faintly superior to ART.

Evaluation of the Training Program to Train HIV Treatment Center Staff in Pakistan.

Introduction In Pakistan, HIV training programs, especially for health professionals working in HIV treatment centers, are limited. Consequently, there is little data about HIV awareness among physicians and allied health workers and how it may affect their care for people living with HIV (PLWH). Recently, the Global Fund to Fight AIDS, Tuberculosis, and Malaria (GFATM) grant Principal Recipient UNDP engaged an NGO experienced in HIV/AIDS training, on a competitive basis, to develop a training manual and conduct training of all categories of HIV treatment centers staff. The goal of this study was to assess the training program's influence on trainees' (both physicians and allied health staff) knowledge and abilities and describe its major lessons. Methodology This was a one-group pre-post test study, carried out between January 17 and February 22, 2023. The study was carried out in three phases. In the first phase, a team of experts developed an antiretroviral treatment (ART) training manual. In the second phase, 9- and three-day training workshops were conducted in six different cities of Pakistan, which were attended by physicians and allied health staff working in different HIV treatment centers across Pakistan. The workshops had plenary lectures, discussions, role plays, video cases, and case studies. In the third phase, a quiz, comprising multiple/best choice questions (MCQs/BCQs) and true and false questions, was administered before (pre) and after the workshop (post) to assess the impact of these training sessions in enhancing the level of HIV knowledge, especially related to ART. The workshop was attended by a total of 256 health workers from different cities in Pakistan. The participants had backgrounds in medical science, psychology, laboratory science, nursing, and computer science. Pre-and post-test responses were statistically analyzed to determine the impact of the training program on participant's knowledge. For this, the Shapiro-Wilk test was applied to test data normality, followed by the application of paired t-test or Wilcoxon Signed Rank Test for normally and non-normally distributed data, respectively. Finally, a chi-square test was applied to examine the significant (p<0.05) association between training workshops and improvement in the participant's level of understanding of HIV. In all statistical tests, p<0.05 was considered significant.  Results The results from our study showed that before the training session, both physicians and allied staff possessed limited knowledge about HIV-related domains. After the workshops, participants from all cities demonstrated a uniform enhancement of knowledge related to different HIV-related domains, evident from the improvement in post-test scores compared to pre-test scores (p<0.0001). The chi-square test showed a significant association between training workshops and improvement in the participant's level of understanding about HIV (p-values for BCQ, MCQ, and true and false: 0.001, 0.0047, and 0.0024, respectively). Conclusions Pre- and post-test evaluation provides an objective, data-driven method for measuring the impact of educational interventions in improving healthcare workers' awareness about HIV. The results emphasize the role of continuous workshops and training programs in enhancing the knowledge and understanding of healthcare and allied health workers regarding HIV.

Impact of gamma irradiation on physico-chemical and electromagnetic interference shielding properties of Cu2O nanoparticles reinforced LDPE nanocomposite films.

In the current work, cuprous oxide (Cu2O) nanoparticles coated with Tween 80 were successfully synthesized via the chemical reduction method. Nanocomposites composed of low-density polyethylene (LDPE) and different ratios of Cu2O nanoparticles were fabricated by the melt mixing process. 10% of ethyl vinyl acetate (EVA) as a compatibilizing agent was added to the molten LDPE matrix and the mixing process continued until homogenous nanocomposites were fabricated. To study the influence of ionizing radiation on the fabricated samples, the prepared species were exposed to 50 and 100 kGy of gamma rays. The synthesized Cu2O nanoparticles were investigated by transmission electron microscopy (TEM) and X-ray diffraction (XRD). XRD and TEM analysis illustrated the successful formation of spherical Cu2O nanoparticles with an average size of 16.8 nm. The as-prepared LDPE/Cu2O nanocomposites were characterized via different techniques such as mechanical, thermal, morphological, XRD, and FTIR. Electromagnetic interference shielding (EMI) of the different nanocomposite formulations was performed as a promising application for these materials in practical life. The electromagnetic shielding effectiveness (SE) of the produced samples was measured in the X-band of the radio frequency range from 8 to 12 GHz using the vector network analyzer (VNA) and a proper waveguide. All the samples were studied before and after gamma-ray irradiation under the same conditions of pressure and temperature. The shielding effectiveness increased significantly from 25 dB for unirradiated samples to 35 dB with samples irradiated with 100 kGy, which reflects 40% enhancement in the effectiveness of the shielding.

Evaluation of colour change and surface roughness of two resin-based composites when exposed to beverages commonly used by children: an in-vitro study.

AIM: To evaluate and compare the surface roughness and colour change of microhybrid and nanocomposite after exposure to beverages commonly used by children. METHODS: Thirty discs were prepared using Filtek Z250 and Filtek Supreme composites. Discs were immersed in distilled water, orange juice and Coca-Cola. Colour and surface roughness were measured at baseline and on days 15 and 30. RESULTS: Filtek Z250 and Filtek Supreme showed no significant difference in surface roughness and colour change at all measurement times. Immersion solution had a significant effect on surface roughness as well as colour change. Specimens immersed in Coca-Cola demonstrated the highest effect, followed by those immersed in orange juice, while specimens immersed in distilled water had the least effect. There was a significant increase in surface roughness and colour change over time. The change in colour was more evident in the period between baseline measurements and after 15 days. There was a significant interaction between media type and time. CONCLUSIONS: Both composites showed no significant difference in roughness and colour change at all measurement times. There was a significant increase in surface roughness and colour change in all immersion solutions tested over time. Coca-Cola caused unacceptable colour change.

Functional and comparative genome analysis of novel virulent actinophages belonging to Streptomyces flavovirens.

BACKGROUND: Next Generation Sequencing (NGS) technologies provide exciting possibilities for whole genome sequencing of a plethora of organisms including bacterial strains and phages, with many possible applications in research and diagnostics. No Streptomyces flavovirens phages have been sequenced to date; there is therefore a lack in available information about S. flavovirens phage genomics. We report biological and physiochemical features and use NGS to provide the complete annotated genomes for two new strains (Sf1 and Sf3) of the virulent phage Streptomyces flavovirens, isolated from Egyptian soil samples. RESULTS: The S. flavovirens phages (Sf1 and Sf3) examined in this study show higher adsorption rates (82 and 85%, respectively) than other actinophages, indicating a strong specificity to their host, and latent periods (15 and 30 min.), followed by rise periods of 45 and 30 min. As expected for actinophages, their burst sizes were 1.95 and 2.49 virions per mL. Both phages were stable and, as reported in previous experiments, showed a significant increase in their activity after sodium chloride (NaCl) and magnesium chloride (MgCl2.6H2O) treatments, whereas after zinc chloride (ZnCl2) application both phages showed a significant decrease in infection. The sequenced phage genomes are parts of a singleton cluster with sizes of 43,150 bp and 60,934 bp, respectively. Bioinformatics analyses and functional characterizations enabled the assignment of possible functions to 19 and 28 putative identified ORFs, which included phage structural proteins, lysis components and metabolic proteins. Thirty phams were identified in both phages, 10 (33.3%) of them with known function, which can be used in cluster prediction. Comparative genomic analysis revealed significant homology between the two phages, showing the highest hits among Sf1, Sf3 and the closest Streptomyces phage (VWB phages) in a specific 13Kb region. However, the phylogenetic analysis using the Major Capsid Protein (MCP) sequences highlighted that the isolated phages belong to the BG Streptomyces phage group but are clearly separated, representing a novel sub-cluster. CONCLUSION: The results of this study provide the first physiological and genomic information for S. flavovirens phages and will be useful for pharmaceutical industries based on S. flavovirens and future phage evolution studies.

A review of traffic safety status in Abu Dhabi city, UAE (2008-2013).

Large numbers of traffic accidents were experienced on the road networks of Arab Gulf Countries including United Arab Emirates (UAE). This had resulted in enormous loss of lives and economy. This article through using Abu Dhabi city, UAE capital as a case study is aiming to understand the reasons behind such safety problem through analysing a large accidents data-set extending over the period from 2008 to 2013. The traffic accidents data-set was obtained from Abu Dhabi traffic police department records and covers wide range of accident's attributes. A wide spectrum of accident attributes was analysed in the paper including but not limited to the time of accident, accident location, type of accident, reasons behind the accident, driver characteristics, road conditions, and many other accident attributes. A specific reasoning for each of these attributes was given by authors. Furthermore, recommendations to enhance the safety levels were introduced.

Risk factors and prevalence of tuberculosis, human immunodeficiency virus, syphilis, hepatitis B virus, and hepatitis C virus among prisoners in Pakistan.

OBJECTIVE: The objective of this study was to evaluate the burden of sexual- and injection drug use-related infections in male prisoners in Karachi, Pakistan. METHODS: We administered a structured questionnaire in a cross-sectional survey of 365 randomly selected imprisoned men. We analyzed blood for the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) by ELISA, and for syphilis by rapid plasma reagin with Treponema pallidum hemagglutination assay confirmation. Subjects with possible tuberculosis (World Health Organization criteria) provided sputum samples for an acid-fast bacillus smear and culture. RESULTS: The prevalence of tuberculosis was 2.2% (95% CI 0.71-3.8%). Of 357 of the randomly selected prisoners (eight refused to give blood), 2.0% (95% CI 0.6-3.4) were HIV-infected; syphilis was confirmed in 8.9% (95% CI 6.0-11.8%), HBV in 5.9% (95% CI 3.5-8.3%), and HCV in 15.2% (95% CI 11.7-18.8). By self-report, 59.2% had used any illicit drugs, among whom 11.8% (95% CI 8.5-15.0) had injected drugs. The median length of stay in the prison had been 3.2 (range 1-72) months. CONCLUSIONS: All four infections were prevalent among the prisoners in Pakistan. Prisons are excellent venues for infectious disease screening and intervention given the conditions of poverty and drug addiction. Collaboration with community-based health providers is vital for post-discharge planning.

Needs assessment and coping strategies of persons infected with HIV in Egypt.

The aims of this study were to identify the psychosocial and health care needs of persons living with HIV/AIDS and to determine their coping strategies. The study was conducted in 3 governorates of Egypt on 153 people infected with HIV. The data were collected using a structured questionnaire and in-depth interviews. The majority of the people had been forced to change the pattern of their sexual behaviour. Fear of stigmatization and feelings of anxiety, hopelessness and depression were frequently reported, resulting in a negative psychosocial impact on the infected person. Decreasing stigma and providing psycho-social support would help people living with HIV/AIDS to utilize effective ways of coping with the negative sequelae of the infection.

Needs assessment and coping strategies of persons infected with HIV in Egypt

The aims of this study were to identify the psychosocial and health care needs of persons living with HIV/AIDS and to determine their coping strategies. The study was conducted in 3 governorates of Egypt on 153 people infected with HIV. The data were collected using a structured questionnaire and in-depth interviews. The majority of the people had been forced to change the pattern of their sexual behaviour. Fear of stigmatization and feelings of anxiety, hopelessness and depression were frequently reported, resulting in a negative psychosocial impact on the infected person. Decreasing stigma and providing psycho-social support would help people living with HIV/AIDS to utilize effective ways of coping with the negative sequelae of the infection

Effect of uniplant on liver function in Egyptian women with asymptomatic hepatitis B virus infection.

Uniplant is a single 35mm contraceptive implant releasing nomegestrol acetate with an effective life span of one year. This study was undertaken to evaluate the possible effects of three-year use of uniplant on some liver enzymes (SGPT, SGOT and GGT) in Egyptian women and to evaluate whether the past asymptomatic hepatitis B virus infection would predispose to any changes in these enzymes. This is an uncontrolled prospective study including 187 women of reproductive age who desired contraception for three years (three segments each year). They were apparently healthy on clinical assessment with no history of jaundice or liver diseases. They also had normal levels of liver function enzymes (SGPT, SGOT & GGT). Fasting blood samples were drawn at admission for assessing the levels of these liver enzymes and to detect anti-HBS antibodies as a marker of past asymptomatic hepatitis B virus infection (HBV) and HBS antigen to diagnose the chronic carrier state. The liver enzymes were measured at 6th. 12th, 18th, 24th, 30th and 36th month of use, and 3-6 months after implant removal. Out of 187 subjects enrolled 159, 83 and 33 women completed one, two and three years of Uniplant use respectively. The mean levels of SGPT, SGOT and GGT showed slight but significant elevations at the end of the first and second years of use compared to the pre-insertion levels. By the end of the third year, the elevated mean values of all enzymes gradually decreased to approach the pre-insertion levels without any significant differences. The percentage of anti-HBS antibodies positive titre due to past asymptomatic infection was 23.3% (n = 41) while HBsAg positive antigenaemia (i.e., carrier state) was 3.4%, (n = 5). In both groups of women--anti-HBS antibodies positive (n = 41) without antigenaemia and anti-HBS antibodies negative (n = 129)--the changes in the mean levels of all enzymes during uniplant use were closely similar without any statistical significance. These results demonstrated that: (1) uniplant induced slight but significant elevations in the mean levels of SGPT, SGOT and GGT these elevations were within the normal ranges, completely reversible and of doubtful clinical significance. (2) The non-carrier women who had been exposed to past asymptomatic HBV infection showed no significant effects on the studied liver enzymes compared to the normal women during long-term use of Uniplant.

Milia en plaque: a new site.

A 35-year-old Kuwaiti field worker presented with a history of an asymptomatic, erythematous plaque on the right side of the nasal bridge. It soon extended to the malar area, being studded with multiple yellowish papules (Fig. 1). He denied any history of photosensitivity, drug intake, local trauma, topical applications, or ionizing radiations to that area. Examination revealed an erythematous, 1.5 x 3 cm plaque on the right nasal fold, extending to the malar area, overlain by a group of tiny yellowish papules (15-20 in number). He also had a few discrete milia on the right cheek. The histology (Fig. 2) revealed multiple keratin-filled cysts, surrounded by a dense lymphocytic infiltrate, findings consistent with milia; 0.05% tretinoin was prescribed twice daily for 1 month without improvement; minocycline, 100 mg daily, was then employed, and at 1 month of follow-up there was a significant decrease in erythema and milia count.

Circulating soluble adhesion molecules in patients with systemic sclerosis: correlation between circulating soluble vascular cell adhesion molecule-1 (sVCAM-1) and impaired left ventricular diastolic function.

The objective of this paper is to investigate the relation between circulating soluble adhesion molecules and cardiac involvement, as assessed by echocardiography in patients with systemic sclerosis (SSc). Nineteen patients with SSc were submitted for assessment of serum levels of circulating soluble intercellular adhesion molecules (sICAM-1), and soluble vascular cell adhesion molecules-1 (sVCAM-1), and echocardiography. Abnormal left ventricular filling patterns (down E/A ratio) were detected in ten patients (52.6%) with significant negative correlation with sVCAM-1 (r = -0.484, P < 0.05). It was also significantly correlated with age (r = -0.791, P < 0.01), age of onset (r = -0.468, P < 0.05), degree of dyspnea (r = -0.687, P < 0.01), and erythrocyte sedimentation rate (ESR) (r = -0.489, P < 0.05). Our findings suggest an important role for sVCAM-1 as a marker of disease severity and impaired left ventricular filling pattern in SSc.

Evaluation of mandibular infiltration versus block anesthesia in pediatric dentistry.

The clinical effectiveness of mandibular block anesthesia was compared to that of buccal infiltration anesthesia. A total of eighty patients three to nine years old was selected with identical bilateral lesions. The anesthetic used was mepacaine HCL 2 percent. The treatments performed were restorations, pulpotomies, and extractions. Child behavior and pain reaction were recorded and rated at certain intervals of treatment, using the Frankl behavioral scale and the SEM scale. The Eland color scale was also used in another sample of twenty patients to determine which type of anesthesia was more acceptable to children. The paired t-test was used to compare results. Buccal infiltration anesthesia was found to be as effective as block anesthesia in all situations, except when pulpotomies were performed in the mandibular second primary molar, where it proved to be unreliable regardless of age. Block anesthesia was significantly more painful than buccal infiltration anesthesia, and behavior of children three through five years old sometimes turned negative following the block injection.

SR-Ca2+ ATPase as an autoimmunogen in experimental myocarditis.

The concept of autoimmunity in the pathogenesis of myocarditis or dilated cardiomyopathy is gaining impetus. Since systolic functional impairment and subsequent recovery are frequently observed in myocarditis, we reasoned that the development of autoimmunity to cardiac sarcoplasmic reticulum calcium ATPase (SR-Ca2+ ATPase), which could interfere with intracellular calcium regulation and therefore affect myocardial contractility, should lead to immune-mediated myocarditis in experimental animals. Murine monoclonal antibody 4C11-20.21 (IgM class) generated against canine cardiac SR-Ca2+ ATPase inhibits the cardiac but not the skeletal ATPase activity. Immunization of CAF1/J mice with 4C11-20.21-affinity-column-purified cardiac SR-ATPase produced a time-dependent induction of myocardial injury consistent with the diagnosis of myocarditis. Furthermore, the antibody 4C11-20.21 alone can induce myo-necrosis in severe combined immunodeficiency (SCID) mice indicating a mechanism of cardiomyopathy independent of the cytotoxic T-cell mediated autoimmunopathy. Administration of 4C11-20.21 into immunocompetent CAF1/J mice resulted in minimal myocardial abnormality (40% with perivascular and/or interstitial mononuclear lymphoplasmacytoid aggregates, 10% with borderline myocarditis and 10% with lesions consistent with focal myocarditis). All control animals had normal hearts. Immunoperoxidase electron microscopic examination of the involved cardiac tissues showed antibody localization in the subsarcolemmal myotubular system and focal staining of the immediately adjacent sarcolemma in mice injected with 4C11-20.21 but not with 2C12.1B5. The time-dependent association between cardiac SR-Ca2+ ATPase administration and development of myocardial lesions, as well as potentiated induction of myonecrosis with anti-cardiac SR-Ca2+ ATPase antibody in SCID relative to immunocompetent mice, suggest a potential autoimmunopathogenic role of cardiac SR-Ca2+ ATPase in experimental myocarditis.

Effect of two types of composite resin on oral bacterial plaque. "In vitro qualitative and quantitative study".

160 specimens of composite (80 chemical and 80 light cured) were prepared in teflon mold 4 mm x 2.5 mm in dimension. Plaque samples were collected using absorbent paper points from 20 normal persons and 20 persons with cervical carious teeth. The samples were inoculated in TSB containing both types of composites. Duplicate cultures were done for aerobic and anaerobic incubations at 37 degrees C. There was no effect of both types of composite on the quality of the isolated organisms from normal group or patient group. Chemical and light cured composite increased significantly the count of aerobic bacterial plaque from normal group. While they decreased the count of aerobic bacterial plaque from patient group and also the anaerobic bacterial plaque from both normal and patient group.

Cardiac sarcoplasmic reticulum calcium ATPase, an autoimmune antigen in experimental cardiomyopathy.

BACKGROUND: Various myocardial cell surface and intracellular antigens have been associated with autoimmune myocarditis. Since sarcoplasmic calcium overload is a recognized pathobiochemical finding in cardiomyopathy, we reasoned that there might be a causal relation between inhibition of sarcoplasmic calcium exclusion and pathogenesis of the disease and that immunization with sarcoplasmic reticulum calcium ATPase (SR-ATPase) or antibody specific for SR-ATPase, which can interfere with the regulation of the intracellular calcium content and the myocardial contractility, should lead to the development of cardiomyopathy and possibly myocarditis. METHODS AND RESULTS: Monoclonal antibody 4C11-20.21 (IgM class) specific for canine cardiac SR-ATPase (M(r) approximately 110 kD) was generated by immunization of CAF1/J mice with dog heart sarcoplasmic reticulum. Antibody 4C11-20.21 inhibits 75% of the enzymatic activity of the cardiac SR-ATPase. This antibody also cross-reacts with the higher M(r) subunit of canine skeletal SR-ATPase, but the skeletal muscle SR-ATPase activity is unaffected. This antibody does not cross-react with sarcolemmal calcium ATPase (134 kD). Antibody 4C11-20.21 was used for affinity purification of cardiac muscle SR-ATPase, which did not contain sarcolemmal calcium ATPase antigen. Nine of 11 CAF1/J mice injected with purified canine cardiac SR-ATPase protein demonstrated myocardial lesions: 3 of 4 mice had occasional perivascular and/or interstitial mononuclear cell infiltrates after 3 weeks, 3 of 4 had borderline myocarditis after 6 weeks, and 3 of 3 had focal myocarditis after 12 weeks. No mononuclear infiltrates were seen in any other organ. To identify the independent effect of 4C11-20.21 antibody on cardiac muscle, 2 x 10(6) hybridoma cells producing the antibody were injected intraperitoneally into 12 severe combined immunodeficiency (SCID) mice. Eleven of 12 SCID mice showed variable cardiac myocyte degeneration without cellular infiltration between 8 and 19 days. Three control SCID mice, which received equivalent injections of hybridoma cells producing IgM anti-myosin light chain antibody, did not show any pathological lesions. Immunoperoxidase staining and/or immunoperoxidase transmission electron microscopy for detection of in vivo localization of 4C11-20.21 demonstrated staining of the subsarcolemmal myotubular system and focal staining of immediately adjacent sarcolemma in animals that received either 4C11-20.21 hybridoma cells or purified canine cardiac SR-ATPase antigen but not in controls. Immunofluorescence staining with goat anti-mouse C3 antibody revealed focal deposition of complement in the cardiac myocytes. CONCLUSIONS: The time-dependent association between immunization with SR-ATPase antigen and the development of myocarditis in mice suggests that cardiac SR-ATPase constitutes one of several autoimmunogens capable of inducing autoimmune myocarditis. Besides antigenic specificity, since antibody to cardiac SR-ATPase also inhibits energy-dependent processes in the myocardium, it is reasonable to associate the pathological evidence of myonecrosis with the interference of calcium regulation, which controls myocardial contractility.

Effect of chlormequat and alar on some biochemical constituents in tomato plants and fruits.

The field experiment was conducted to study the effect of various levels of chlormequat (CCC) and alar on the biochemical changes in tomato plants and fruits at different stages of growth. This experiment included spraying with chlormequat and alar separately in two equal doses (250, 500 and 1000 ppm CCC or alar 25 and 40 days after transplanting). The different levels of chlormequat decreased the accumulation of dry matter in tomato plants, but alar increased it. Chlorophyll a, b, total chlorophyll and carotenoids content of tomato plants increased by the application of CCC or alar. The highest increase of concentration of chlorophyll a, b and carotenoids in tomato plants were found by spraying with 500 ppm alar or CCC. The application of CCC and alar declined the percentage of carbohydrates and the highest decrease resulted by adding of 1000 ppm alar or CCC. Alar caused an increase in the percentage of total nitrogen at the different stages of growth. The concentration of P, K, Ca and Mg increased by the foliar spray of all treatments. Alar application at all used levels significantly increased the yield and also the weight of fruits. Highest plant productivity was obtained by using alar and CCC at 250 ppm, followed by 500 ppm. However, the highest concentration (1000 ppm) depressed the plant productivity. The concentration of juice, total soluble solids and vitamin C in tomato fruits increased at most of the levels added. But the percentage of total sugars and total acidity seemed to exert another trend. The highest concentration of N, P, K, Ca and Mg in fruits was obtained by foliar application of 500 ppm CCC or alar.

Pharmacokinetics of diclofenac sodium using a developed HPLC method.

The pharmacokinetics of 2-[(2,6-dichlorophenyl)-amino]phenyl acetic acid (diclofenac sodium) was studied after a single oral dose of 10 mg/kg in rabbits, using a developed HPLC method. The observed serum levels of rabbits were fitted to a one-compartment open model, with first-order absorption and elimination. The mean values of t1/2e, ke, 51/2a, ka and tmax were 1.98 h, 0.3455 h-1, 1.2357 h, 1.2357 h, 0.5653 h-1 and 2.2267 h, respectively. This high-performance liquid chromatographic method for the rapid (about 10 min), sensitive (1 microgram/ml) and specific determination of diclofenac sodium in serum and urine was developed using acetaminophen as the internal standard, a reversed-phase C 18 column, and ethanol: water (1:2) as the mobile phase.