Development of a Method for Identification and Quantification of Sulfadiazine and Pyrimethamine in Serum of Congenital Toxoplasmosis Pediatric Patients

Infection with Toxoplasma gondii, is one of the most widespread zoonoses in the world. Congenital Toxoplasmosis (CT) is particularly risky due to its fetal complications. Sulfadiazine (SDZ) and Pyrimethamine (PYR) are usually used for CT treatment in Argentina, to prevent morbidity. Due to the lack of com-mercial pediatric formulations, these must (<LOD - 162.04 ± 0.02) µg/mL for SDZ and (<LOD - 7.30 ± 0.03) µg/mL for PYR. We developed and validated in real pediatric samples, an acute, precise and low-cost method for quantification of SDZ and PYR using a non-sophisti-cate chromatographic equipment, suitable for hospital therapeutic monitoring for public health system.


Introduction
Infection with Toxoplasma gondii, is one of the most widespread zoonoses in the world. The worldwide infection prevalence is estimated at 30%. The infection is acquired mainly by food contaminated with parasite cysts, and is usually asymptomatic. The congenital infection is particularly risky, with rates of mother-to-baby transmission approaching 90% in the last month of pregnancy. There are few studies on the incidence of Congenital Toxoplasmosis (CT) in Latin America, but the seroprevalence in women of childbearing age is high [1]. Mostly the infection is asymptomatic, and the diagnosis is made by serological screening. Treatment during pregnancy decreased fetal morbidity and sequelae in the child [2]. An early diagnosis followed by treatment of CT in infants provides a better resolution of clinical signs compared to those not treated [3] [4]. Between 10% and 30% of prenatal infections result in abortion, death of the newborn or severe clinical signs at birth [5] [6]. However, about 67% of congenital infections are clinically asymptomatic at birth and may develop symptoms later, predominantly ocular lesions [3] [6].
The current therapy in pediatric patients is protocolized, but due to the absence of pediatric formulations of the drugs, these are prepared in the hospital pharmacy in the form of syrup and at the moment, pharmacological parameters of these drugs have not been corroborated on serum samples for this combination of SDZ and PYR.
Drugs available for the treatment of toxoplasmosis only inhibit the growth of the parasite when it is in the active phase of its life cycle (tachyzoite), not being useful against the cystic or latent form of the parasite (bradyzoites). Most health centers do not hesitate to recommend treatment to infants with confirmed CT. However, to date, there is no controlled study in our country that determines its efficacy, the appropriate therapeutic dose and the optimal duration [6].
Indeed, there are some coincidences about the drugs to be used, but the duration of treatment has been more discussed. Prolonged treatments are associated with a lower rate of sequelae while short treatments have the advantage of reducing drug toxicity. The treatment scheme in Argentina is SDZ 50 -100 mg/kg/d associated with PYR 1 mg/kg/d and folinic acid 5 mg/48 hours. The duration is from diagnosis to one year of age with a minimum time of 6 months if the child is older. PYR (5-(4-Chlorophenyl)-6-ethyl-2,4-diaminopyrimidine) interferes with the synthesis of folic acid by inhibiting dihydropteroate synthase and dihydrofolate reductase and due to poorly studied pharmacological factors, treatment may not be successful.
SDZ (N-amino-N-pyrimidin-2-yl-benzenesulfonamide) is the most active sulfamide against T. gondii. It has synergistic activity with PYR but being analogous to the PABA, necessary for the production of parasitic nucleic acids. It is excreted by the kidney, requiring dose adjustment in patients with renal impairment. It is not indicated in patients with glucose deficiency 6-phosphate dehydrogenase (G6PDH) and replaced with clindamycin.

Methods
A protocol designed to evaluate the response of a bioanalytical method for identification and simultaneous quantification of SDZ and PYR by High Performance Liquid Chromatography (HPLC) with UV detection was followed. The aim is to validate the HPLC-UV method in order to transfer these capabilities to health institutions that perform therapeutic monitoring of these drugs. Instrumental techniques using HPLC-UV require equipment of medium complexity suitable for the monitoring of pharmacotherapy, available in hospitals and institutions of the public health system in Argentina.

SDZ and PYR Stability over Time
Accurate standard solutions of SDZ, PYR and a mix of both of them were eva-luated at different concentration levels. Duplicate samples of these standard solutions were stored in batches in refrigerator and freezer, respectively. After a defined period of time (between 24 hours and 30 days of storage) each solution was quantified. For every sample, its percentage coefficient of variation (CV%) intra-day (repeatability) and inter-day (reproducibility) was determined after its storage in refrigerator or freezer. Here, there was no pretreatment step needed for these samples, so as to evaluate only the chromatographic system response and the stability of these standard solutions.
An analogous storage treatment was performed for spiked serum samples at accurate and known concentrations of SDZ, PYR and a mix of both. Stability concentration after storage in the freezer between 24 hours and 30 days was also evaluated for these samples. Here, a pretreatment was needed to extract SDZ and PYR from the serum matrix. Percentage of Recovery (R%) was calculated for all stored samples and for a fresh spiked serum sample with an accurate concentration as one of those stored, in every day of analysis.
Finally, another stability evaluation was performed in 4 serum samples after 3 cycles of defreeze/freeze at an accurate and known concentrations of SDZ, PYR and a mix of both, followed by extraction and quantification, according to protocols proposed by the National Administration of Medicines, Food and Medical Technology (ANMAT) for the stability of a bioanalytical method [11]. It was proposed as a stablity criterion those samples that presented a CV% ≤ 15.0% in all instances.

Materials and Reagents
Trichloroacetic Acid (TCA) pro analysis grade was purchased from Biopack (Buenos Aires, Argentina). Dimethyl Sulfoxide (DMSO) pro analysis grade was obtained from Anedra (Buenos Aires, Argentina). Chromatographic grade demineralized water (<0.2 μsiemens) was obtained in our laboratory with ionic interchange resins. Acetonitrile (AcN) and Methanol (Me) (J.T. Baker, USA) HPLCgrade were used. Sulfadiazine (Stanton L_1205050015269/0088) was obtained from the hospital pharmacy and pyrimethamine was obtained from sigma Aldrich.
A Table Top  was also used on extraction procedures.

HPLC Instrumentation and Calculation
The instrumental analytical procedure for samples measures were performed  (Table   1).
Also, variable volumes of these standard solutions were added to drug-free serum to obtain matrix standards of 1000 µL volume for calibration curves. These  (Table 2). In all cases the volume of the standard solution added to the serum matrix did not exceed 20% off its volume to minimize dilution effects. For control specimen a 20% serum sample volume of the solvent used for standard solutions was added.

Serum Samples
Residual serum samples were obtained from six pediatric patients treated for TC from a clinical study with PYR (0.77 a 2.70 mg/kg/day), aged between 33 days and 3 year old. Samples were stored at −20˚C until analysis.
The clinical study and its informed consent for the use of the samples were approved by the institutional ethics committee of the Ricardo Gutiérrez Children's Hospital (RGCH).

Sample Pretreatment and Chromatographic Conditions
All 1000 μL samples were deproteinized with 50 μL of TCA (30% p/v), vortexed for one minute, and sonicated for five minutes. The mixture was then centrifuged at 8000 g for another five 5 min. After this, 300 μL of the supernatant were separated on an eppendorf before injecting it into the HPLC system.
The HPLC analysis was performed by a gradient elution in a Reverse Phase (RP) mode. The mobile phase composition varied from 90% to 50% of water (1% v/v of formic acid) with methanol from 5% to 45% and 5% of acetonitrile that remained constant throughout the run. The flow ranged was 0.8 to 1.0 mL/min and the total running time was fourteen minutes. All solvents were filtered through a 0.45 μm nylon membrane and degassed before use. The maximum UV absorption found for simultaneous identification of SDZ and PYR was at 273 nm, so this wavelength was chosen for the method. A value of 0.030 absorbance units (a.u.) threshold was used. Duplicate injections were made for all samples to test reproducibility of the detector response at each concentration level. Peak area was plotted against concentration to obtain calibration graphs. Linear regression and a one way analysis of variance were applied to calculate calibration equation and statistical correlation coefficients. Table 3 and Figure 1 show intraday CV% (repeatability), between duplicates of standard solutions in refrigerator and freezer after each storage time. The last two columns of the table present the CV% inter-day (reproducibility) for each standard in solvent or in serum for storage in refrigerator or freezer respectively.

Results
Higher concentrated standard solutions presented lower CV% than those diluted. Although the reproducibility in no case exceeds the maximum CV% proposed as acceptable, it was found that the standard solutions stored in the refrigerator within the period evaluated, had lower CV% than those stored in the freezer. This difference may be due to factors such as the decrease in the solubility of drugs at low temperatures associated with some systematic error in the homogenization prior to injection into the chromatographic equipment.
No evaluation on serum was made for sample storage in the refrigerator because it is well known that serum samples refrigerator storage is not recommended for periods longer than 24 hours. For this reason, matrix standard solutions storage behavior were only studied in freezer. Here, there were no significant  differences in CV% or R% between high or low concentrations of both SDZ and PYR. Also, the presence of the two drugs together in the matrix did not cause analytical interference or significant increases in its CV% or decreases on R%. The CV% obtained for serum samples after 3 cycles of defrosting/freezing are presented in Table 4. There, it can be observed that the CV% are slightly higher for PYR than for SDZ (average CV% of 12.88 for the SDZ and 14.03 for the PYR) but they do not exceed the values proposed as acceptable in any case.
Calibration curves for simultaneous detection and quantification of SDZ and PYR were made on three different days with six concentration levels. Total retention time range for gradient mode used on chromatographic analysis was (5.01 -5.36) minutes for SDZ and (12.16 -12.60) minutes for PYR. Also, SDZ and PYR retention time ratio had a range of 0.41 -0.43 and a total run time was 14 minutes per sample. The LOD and LOQ were (0.17 ± 0.02 and 0.13 ± 0.02) µg/mL and (0.46 ± 0.01 and 0.36 ± 0.01) µg/mL for SDZ and PYR respectively. The validated method had a linear range of (<LOQ -210.00 ± 0.02) µg/mL for SDZ and (<LOQ -15.05 ± 0.02) µg/mL for PYR. A summary of calibration curve statistical parameters are presented on Table 5.
Chromatographic parameters such as resolution, selectivity, and peak asymmetry were satisfactory for SDZ and PYR determination with this method. In addition, analytes showed no decomposition products detectable in chromatograms profiles. Figure 2 presents a characteristic chromatogram profile for the proposed RP gradient chromatographic system for the detection and quantification of SDZ and PYR in (a) standard solutions mix (b) serum extracts for SDZ and PYR.

SDZ and PYR on Pediatric Samples
With the validated method, SDZ and PYR were measured in a set of 30 samples from 6 pediatric patients who participated in a clinical study to evaluate the pharmacokinetics of both drugs on serum samples. The concentrations range

Discussion
The two drugs analytically evaluated on this study (SDZ and PYR), are currently available for the treatment of TC on public health Institutions in Argentina. However, serum concentrations coming from pediatric formulations prepared in hospital pharmacy have not been quantified before. Until now, there were no validated methods by HPLC/UV developed for the simultaneous detection of these drugs nor doses. In this sense, it was possible to corroborate stability of these drugs on different matrices and its dependence on sample storage period between refrigerator and freezer. This proves the robustness needed for a method suitable for therapeutic monitoring, pharmacokinetics or toxicokinetics. In literature, there is a developed method for determination of PYR, sulfadoxine, mefloquine, and ibuprofen by HPLC/UV for determination of these drugs in raw materials and dosage of pharmaceutical formulations but SDZ was not included [12]. Also, there are methods developed for SDZ and its hydroxy metabolite and its quantification by reverse phase HPLC [13], and there are others for PYR by HPLC and fluorescence detection, but applied to the malaria pharmacotherapy [14] or for TC but with sulfadoxine instead of SDZ also by HPLC/UV.
There is an interest in the determination of clinically significant serum range of SDZ and PYR concentrations. To our knowledge, there are two different scenarios: adult serum concentration and pediatric serum concentrations. The LOQ of the method described here for both SDZ and PYR, seems to be appropriate in pediatric contexts. Most of the pediatric samples obtained were of 1.00 mL or less volume of serum. In this sense, minor sample volume may imply a decrease in the sensibility of the method, so there is a compromise between these two variables, also attending that blood samples volumes in pediatrics are normally smaller than in adults. In this sense, it is important to note that pediatric samples analysed for validation procedures were not specially taken. Instead of that, residual volumes of serum were used from an ongoing clinical study.
The development of HPLC methods for determination of drugs has received considerate attention in recent years because of their importance in the quality control of drugs and drug products. The goal of this study was to develop an accurate, precise, and low cost HPLC method for quantification of SDZ and PYR simultaneously, using the most commonly employed C-18 column with UV detection.
Instrumental techniques using HPLC-UV require equipment of medium complexity suitable for the monitoring of pharmacotherapy, available in hospitals and institutions of the public health system, giving its advantages over others reported HPLC methods for determination of some of these two drugs but through more expensive and sophisticated detection systems [15].

Conclusions
We developed and validated in real pediatric samples, an acute, precise and low-cost method for quantification of SDZ and PYR using a non-sophisticate chromatographic equipment, suitable for hospital therapeutic monitoring for public health system. This is the first report of serum concentrations measures in pediatric CT therapy for this combination of drugs.

Funding and Acknowledgements
The authors would like to gratefully acknowledge the financial support and the doctoral and postdoctoral scholarships received from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and Comisión de Investigaciones Científicas de la provincia de Buenos Aires (CICpBA).

Compliance with Ethical Standards
The clinical study protocol and its informed consent for the use of human samples were approved by the Institutional Ethics Committee of the Ricardo Gutiérrez Children's Hospital.