N.P.J. Day

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of piperaquine in plasma Stable isotope labeled internal standard does not always compensate for matrix effects

A bioanalytical method for the analysis of piperaquine in human plasma using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. It was found that a mobile phase with high pH (i.e. 10) led to better sensitivity than mobile phase combinations with low pH (i.e. 2.5-4.5) despite the use of positive electrospray and a basic analyte. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as R.S.D., were lower than 7% at all tested concentrations (4.5, 20, 400 and 500ng/mL) and below 10% at the lower limit of quantification (LLOQ) (1.5ng/mL). The calibration range was 1.5-500ng/mL with a limit of detection (LOD) at 0.38ng/mL. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. Matrix effects originating from the sample clean-up (i.e. solid-phase extraction) procedure rather than the plasma background were responsible for the ion suppression seen in this study. Salts remaining from the buffers used in the solid-phase extraction suppressed the signals for both piperaquine and its deuterated internal standard. This had no effect on the quantification of piperaquine. Triethylamine residues remaining after evaporation of the solid-phase extraction eluate were found to suppress the signals for piperaquine and its deuterated internal standard differently. It was found that this could lead to an underestimation of the true concentration with 50% despite the use of a deuterated internal standard.

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma.

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Neuropathological assessment of artemether-treated severe malaria

In animals, high doses of intramuscular artemether and artemotil have been shown to cause an unusual pattern of selective damage to certain brainstem nuclei, especially those implicated in hearing and balance. We aimed to investigate whether a similar pattern arises in human adults. We examined the brainstems of adults who died after treatment with high dose artemether or quinine for severe falciparum malaria for evidence of a pattern of selective neuronal damage. Neuropathological findings were similar in recipients of quinine (n=15) and artemether (n=6; total artemether doses received 4-44 mg/kg). No evidence was recorded for artemether-induced neurotoxic effects.

Characterization of Human Urinary Metabolites of the Antimalarial Piperaquine

Five metabolites of the antimalarial piperaquine (PQ) (1,3-bis-[4-(7-chloroquinolyl-4)-piperazinyl-1]-propane) have been identified and their molecular structures characterized. After a p.o. dose of dihydroartemisinin-piperaquine, urine collected over 16 h from two healthy subjects was analyzed using liquid chromatography (LC)/UV, LC/tandem mass spectrometry (MS/MS), Fourier transform ion cyclotron resonance (FTICR)/MS, and H NMR. Five different peaks were recognized as possible metabolites [M1, 320 m/z; M2, M3, and M4, 551 m/z (PQ + 16 m/z); and M5, 567 m/z (PQ + 32 m/z)] using LC/MS/MS with gradient elution. The proposed carboxylic M1 has a theoretical monoisotopic molecular mass of 320.1166 m/z, which is in accordance with the FTICR/MS (320.1168 m/z) findings. The LC/MS/MS results also showed a 551 m/z metabolite (M2) with a distinct difference both in polarity and fragmentation pattern compared with PQ, 7-hydroxypiperaquine, and the other 551 m/z metabolites. We suggest that this is caused by N-oxidation of PQ. The results showed two metabolites (M3 and M4) with a molecular ion at 551 m/z and similar fragmentation pattern as both PQ and 7-hydroxypiperaquine; therefore, they are likely to be hydroxylated PQ metabolites. The molecular structures of M1 and M2 were also confirmed using H NMR. Urinary excretion rate in one subject suggested a terminal elimination half-life of about 53 days for M1. Assuming formation rate-limiting kinetics, this would support recent findings that the terminal elimination half-life of PQ has been underestimated previously.

Validation and application of a liquid chromatographic–mass spectrometric method for determination of artesunate in pharmaceutical samples

A simple and rapid liquid chromatographic-mass spectrometric assay for the evaluation of artesunate in vials for injection has been developed and validated. The content of each vial was dissolved in 3.0 mL of methanol using a SGE analytical syringe (1.0 mL). Each sample was diluted to a theoretical concentration of 1000 ng/mL and analysed in triplicate. Three replicates of calibration standards at concentrations 500, 1000 and 1500 ng/mL were used to construct a calibration curve. Artesunate was analysed by liquid chromatography with atmospheric pressure chemical ionisation (APCI) mass spectrometric (MS) detection on a Hypersil Gold column (100 mm x 4.6 mm) using a mobile phase containing methanol-ammonium acetate 10 mM pH 5.3 (70:30, v/v) at a flow rate of 1 mL/min. The assay was implemented for the analysis of artesunate for injection purchased from Guilin Pharmaceutical Company in China.

Development and validation of a high-throughput zwitterionic hydrophilic interaction liquid chromatography solid-phase extraction–liquid chromatography–tandem mass spectrometry method for determination of the anti-influenza drug peramivir in plasma

An assay for the analysis for the quantification of the anti-influenza drug peramivir in human plasma using high-throughput zwitterionic (ZIC) hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) in a 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The ZIC-HILIC SPE efficiently removed sources of interference present in the supernatant after protein precipitation of plasma proteins. The main advantage of the ZIC-HILIC SPE sample preparation step was that it allowed load and elution conditions to be optimised to extract only peramivir and minimize co-extraction of lipophilic phospholipids. The method was validated according to published US Food and Drugs Administration guidelines and showed excellent performance. The assay was validated over two calibration ranges (0.952-500 and 50-50,000 ng/mL) to support analysis of peramivir after intra-venous administration. The lower limit of quantification for peramivir in plasma was 1 ng/mL and the upper limit of quantification was 50,000 ng/mL. The within-day and between-day precisions expressed as RSD, were lower than 8% at all tested quality control concentrations and below 11% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range.

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Predicting the clinical outcome of tetanus : the tetanus severity score

Objectives  To create a new tetanus score and compare it with the Phillips and Dakar scores.

The value of intermittent point-prevalence surveys of healthcare-associated infections for evaluating infection control interventions at Angkor Hospital for Children, Siem Reap, Cambodia

Background There are limited data on the epidemiology of paediatric healthcare-associated infection (HCAI) and infection control in low-income countries. We describe the value of intermittent point-prevalence surveys for monitoring HCAI and evaluating infection control interventions in a Cambodian paediatric hospital. Methods Hospital-wide, point-prevalence surveys were performed monthly in 2011. Infection control interventions introduced during this period included a hand hygiene programme and a ventilator-associated pneumonia (VAP) care bundle. Results Overall HCAI prevalence was 13.8/100 patients at-risk, with a significant decline over time. The highest HCAI rates (50%) were observed in critical care; the majority of HCAIs were respiratory (61%). Klebsiella pneumoniae was most commonly isolated and antimicrobial resistance was widespread. Hand hygiene compliance doubled to 51.6%, and total VAP cases/1000 patient-ventilator days fell from 30 to 10. Conclusion Rates of HCAI were substantial in our institution, and antimicrobial resistance a major concern. Point-prevalence surveys are effective for HCAI surveillance, and in monitoring trends in response to infection control interventions.

Cost-effectiveness of hand hygiene promotion for MRSA blood stream infection in ICU settings

Multimodal interventions are effective in increasing hand hygiene compliance amongst healthcare workers, but it is not known whether such interventions are cost-effective outside high-income countries.