Analytical Investigations of Toxic p-Phenylenediamine (PPD) Levels in Clinical Urine Samples with Special Focus on MALDI-MS/MS

Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N′-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (∼10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (∼25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50–1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples

The Impact of Virtual Learning on EEL Learners’ Performance in the Assessment Results during Covid19 Pandemic

In early 2020, a sudden pandemic known later as Covid19 spread all over the world and in the Gulf region. The immediate action of education authorities in the Gulf was taken after a short break period to shift the learning system across the Gulf from conventional face-to-face learning to virtual learning. This paper investigated the impact of the sudden shift in learning from the institution students' feedback and academic performance during the COVID-19 epidemic study period. This study intends to analyze the impact of virtual learning on EEL learners' performance within the assessment results during the Covid-19 pandemic and also to assess the usage of the assessment results from tools at the time of the Covid19 pandemic. Moreover, it intends to give the students' educational performance once the sudden shift into Virtual- Learning supported their final ends up results in the assessment results. The information was gathered via a questionnaire which was distributed randomly among English male students in the English Group Centre. It was responded to by 23 students. Items of the questionnaire were designed quantitatively. The organized inquiries estimated the assessment reactions to explain the target reactions and simultaneously improve the definition of suggestions of the study. The study recommends making students well-trained in the electronic assessment mode so as to promote the learners’ performance within the assessment results online since the result indicated that they prefer traditional learning to Virtual learning

Validation results of PPD in human urine.

<p>MALDI-MS/MS assay: linearity, lower limit of quantification (LLOQ), recovery (at 130, 400 and 800 µmol/L) and stability after addition of formic acid.</p

Validation results of PPD in human urine.

<p>Inter- and intraday accuracy and precision measurements (<i>n = 3</i> for each value). The mean relative error of each concentration is expressed as % bias and the reproducibility is depicted as the coefficient of variation (% CV). Intraday accuracy and precision were also determined on the HPLC-UV system.</p

Calibration curve of PPD (A) on the MALDI-MS/MS system (50–1000 µmol/L) and (B) on the HPLC-UV system (150–1000 µmol/L).

<p>Graphs shows the mean values of (<b>A</b>) 5 and (<b>B</b>) 3 independent measurements and the corresponding <i>r²</i> value. Mean +/− SEM.</p

MALDI-MS/MS measurements of urine samples from patients with suspected PPD intoxication and controls.

<p>(<b>A</b>) Measured PPD concentrations in clinical urine samples (<i>n = 15</i>); dotted lines show the LLOQ of the MALDI-MS/MS and the HPLC-UV application, resp. (<b>B</b>) Box-and-whisker blot with the 5–95 percentiles for the comparison of PPD, MAPPD and DAPPD peak areas in drug free control urine (<i>n = 5</i>) and clinical samples of intoxication (<i>n = 15</i>). (<b>C</b>) Data correlation (<i>r²</i> = 0.7618) of PPD and DAPPD in patient urine (<i>n = 15</i>). Respective p-values are given in the graphs.</p

Chemical structures and respective multiple reaction monitoring (MRM) transitions of (A) PPD and its metabolites (MAPPD and DAPPD) and (B) the internal standard (IS) 2-amino-5-nitropyridine.

<p>Chemical structures and respective multiple reaction monitoring (MRM) transitions of (A) PPD and its metabolites (MAPPD and DAPPD) and (B) the internal standard (IS) 2-amino-5-nitropyridine.</p

Wet chemistry assay for spiked blank and clinical urine samples.

<p>(<b>A</b>) <i>Left row bottom to top:</i> urine blank and 5 different spiked concentrations (25, 50, 100, 250 and 500 µmol/L). <i>Right row top three vials:</i> three clinical samples, which show a positive test result for PPD. (<b>B</b>) Concentration dependent transmittance (%) from 25–250 µmol/L on a spectrophotometer at 450 nm.</p

TOF spectrum (pos. mode) for the identification of two PPD metabolites MAPPD (<i>m/z</i> [M+H]⁺ 151) and DAPPD (<i>m/z</i> [M+H]⁺ 193) in patient urine.

<p>Comparative control urine did not show any signals at the corresponding <i>m/z</i> values.</p