Development of a Method for Identification and Quantification of Sulfadiazine and Pyrimethamine in Serum of Congenital Toxoplasmosis 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. Global risk of CT transmission is approximately 40%, reaching 90% in the last month of pregnancy. Children with CT frequently require treatment, usually in Argentina with sulfadiazine (SDZ) and pyrimethamine (PYR), to prevent morbidity. Therapy for pediatric patients is hampered by the absence of pediatric formulations. To address this problem, SDZ and PYR are prepared as extemporaneous formulations by hospital pharmacies in the form of syrups. At the moment, serological concentrations of these formulations have not been corroborated in patient serum samples. The objective of this study was to develop a bioanalytical method for identification and simultaneous quantification of SDZ and PYR by high performance liquid chromatography (HPLC) with UV detection. The validated method was tested with residual serum samples obtained from 6 pediatric patients undergoing treatment with SDZ 42.20 a 93.70 mg/kg/day and PYR 0.77 a 2.70 mg/kg/day. Calibration curves were made for SDZ and PYR by spiking both drugs on drug-free serum samples. Pretreatment consisted of a deproteinization step with trichloroacetic acid followed by centrifugation and then injection of supernatant. Limit of detection (LOD) and quantification (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. Serum samples range concentrations found were (<LOD - 162.04 ±0.02) µg/mL for SDZ and (< LOD - 7.30 ±0.03) µg/mL for PYR. We developed a rapid, accurate, precise HPLC method for quantification of SDZ and PYR simultaneously, using the most commonly employed C-18 column with UV detection with sufficient sensitivity to be applied to therapeutic monitoring in pediatrics. It is the first report of dosages of serum concentrations of SDZ and PYR in pediatric samples carried out in public institutions in Argentina.


Introduction
Infection with Toxoplasma gondii, is one of the most widespread zoonoses in the world. It is estimated that one third of the world's population is infected. 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 locally corroborated in this population of patients, especially for the 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 is a coincidence 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)-6ethyl-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-2yl-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.
In other countries there are few studies in the pediatric population where PYR and SDZ on serum samples are quantified but they are framed in populations in which different drugs and combinations of drugs are used with different therapeutic and combination of drugs used [7][8][9]. A publication describing pharmacokinetic parameters for pediatric population treated several months for CT with PYR and sulfadoxine, proposes the existence of a wide interindividual variability and that at a dose adjusted to weight, plasma concentrations would be unpredictable. Therefore, it has not been possible to establish what plasma concentration in the combination of drugs is most effective in pediatrics. The relationship between therapeutic blood concentrations and toxicity is unknown and there are also no studies of interaction with new anticonvulsants or corticosteroids.
On the other hand, the transfer information of these drugs through the placenta or breast milk is scarce. All these vacancies translate into important working hypotheses. Despite addressing drugs with a long time of use in therapeutics, there is no sufficient information in the literature in the pediatric field. To advance in any of the hypotheses, a simple, fast, precise and clinically adjusted method, such as the one presented in this work, is of great importance as a tool for systematization and improvement of the current pharmacological treatment protocols for this disease.

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. 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 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 [10]. 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 to take as a stable criterion those samples that presented a CV % ≤ 15.0 % in all instances. All micropipettes were calibrated before use. All HPLC solvents were degassed with a vacuum pump (Pascal, Buenos Aires, Argentina).

Materials and Reagents
An ultrasonic homogenizer (FAETA, Argentina) was also used on extraction procedures.

HPLC Instrumentation and Calculation
The instrumental analytical procedure for samples measures were performed with an LC system consisting of an HPLC Merck- Accuracy and precision of the assays were calculated based on the analysis of three replicates for each level of the standard curve. Total uncertainty was calculated as the sum of accuracy and precision.

Sample Pretreatment and Chromatographic Conditions
All 1,000μL 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, 300μL of the supernatant were separated on an eppendorf before injecting it into the HPLC system.  Table 3 and Figure 1 shows intraday CV % (repeatability), between duplicates of standard solutions in refrigerator and freezer after each storage time. The last two columns of the 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 %R.  The CV % obtained for serum samples after 3 cycles of defrosting / frizzing are presented in Table 4. There, it can be observed that the CV% are slightly higher for PYR than for SDZ (average CV% of  Table 5.

Results
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

Discussion
The two drugs analytically evaluated on this study SDZ and 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 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].

Conclusion
A rapid, precise, accurate, low-cost, RP-HPLC-UV method for simultaneous identification and quantification of SDZ and

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.