Addition of sulfadoxine- for Intermittent Preventive Treatment in pregnancy in the WHO Model List of Essential Medicines for adults

General items

1. Summary statement of the proposal for inclusion, change or deletion.

This proposal suggests the addition of sulfadoxine-pyrimethamine (SP) for Intermittent Preventive Treatment in pregnancy (IPTp) in the WHO Model List of Essential Medicines (EML) for adults. The objective of preventive treatment is to prevent malarial illness by maintaining therapeutic drug levels in the blood throughout the period of greatest risk. Current WHO-recommended chemopreventive therapies include IPTp, IPTi and SMC.

Malaria infection during pregnancy is a major public health problem, with substantial risks for the mother, her fetus and the newborn. WHO recommends a package of interventions for preventing and controlling malaria during pregnancy, which includes promotion and use of insecticide-treated nets, appropriate case management with prompt, effective treatment and, in areas with moderate to high transmission of P. falciparum, administration of IPTp-SP. The specific WHO recommendation for IPTp with SP reads as follows in the WHO Guidelines for the treatment of malaria:

In malaria-endemic areas in Africa, provide intermittent preventive treatment with SP to all women in their first or second pregnancy (SP-IPTp) as part of antenatal care. Dosing should start in the second trimester and doses should be given at least 1 month apart, with the objective of ensuring that at least three doses are received.

According to the GRADE system, this is a strong recommendation, based on high-quality evidence. (1)

2. Relevant WHO technical department and focal point (if applicable).

WHO Global Malaria Programme (WHO/GMP). Focal Point: Silvia Schwarte, e-mail: [email protected]

3. Name of organization(s) consulted and/or supporting the application.

N/A

4. International Nonproprietary Name (INN), Anatomical Therapeutic Chemical (ATC) code of the medicine and International Classification of Disease (ICD) 11.

. INN: Sulfadoxine-pyrimethamine . ATC: P01BD51 pyrimethamine, combinations (2) . ICD-11: XM90Z4 Pyrimethamine with sulfadoxine (3)

5. Dose forms(s) and strength(s) proposed for inclusion; including adult and paediatric (if appropriate).

Sulfadoxine-pyrimethamine is available as fixed-dose combination tablet at a strength of 500 mg sulfadoxine plus 25 mg pyrimethamine. The required monthly dose in the context of IPTp-SP, i.e. 1500 mg sulfadoxine plus 75 mg pyrimethamine, is 3 tablets of this strength. As the preventive medicines is intended for use in pregnant women, it is suggested for inclusion in the EML for adults.

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6. Whether listing is requested as an individual medicine or as representative of a pharmacological class.

Listing is requested on the Model List of Essential Medicines for adults as an individual medicine, to be included in the section 6.5 medicines under chapter 6.5.3.2 For prophylaxis.

7. Treatment details (requirements for diagnosis, treatment and monitoring).

Summary recommendation. Based on the evidence review described in more detail in section 9 below, in October 2012 WHO updated the recommendations on IPTp-SP, which is an integral part of WHO’s three-pronged approach to the prevention and treatment of malaria in pregnancy, besides the use of insecticide-treated nets and prompt and effective case management. All possible efforts should be made to increase access to IPTp-SP in all areas with moderate to high malaria transmission1 in Africa, as part of antenatal care services:

. Starting as early as possible in the second trimester, IPTp-SP is recommended for all pregnant women at each scheduled antenatal care (ANC) visit until the time of delivery, provided that the doses are given at least one month apart. SP should not be given during the first trimester of pregnancy; however, the last dose of IPTp-SP can be administered up to the time of delivery without safety concerns (4). ‒ IPTp-SP should ideally be administered as directly observed therapy (DOT) of three tablets sulfadoxine/pyrimethamine (each tablet containing 500 mg/25 mg SP) giving the total required dosage of 1500 mg/75 mg SP. ‒ SP can be given either on an empty stomach or with food. ‒ SP should not be administered to women receiving co-trimoxazole prophylaxis due to a higher risk of adverse events. ‒ WHO recommends the administration of folic acid at a dose of 0.4 mg daily; this dose may be safely used in conjunction with SP. Folic acid at a daily dose equal or above 5 mg should not be given together with SP as this counteracts its efficacy as an antimalarial. . In some countries of sub-Saharan Africa, transmission of malaria has been reduced substantially due to the successful implementation of malaria control efforts. In the absence of data to help determine when to stop IPTp-SP, WHO recommends that countries continue to provide IPTp-SP until data to guide this decision-making is available. . There is currently insufficient evidence to support a general recommendation for the use of IPTp-SP outside Africa.

Administration. There is limited evidence of potential teratogenicity when SP is used in the first trimester (4, 5). Thus, and until more safety data becomes available, this medicine should not be used during the first trimester. During these early weeks of pregnancy, a woman should protect herself against malaria by using an insecticide-treated net. IPTp-SP can, however, be administered safely at the beginning of the second trimester, i.e. starting at the beginning of the thirteenth week. In the absence of gestational dating by ultrasound, the beginning of the second trimester can be determined by measuring fundal height which may serve as a proxy for gestational age. The fundal height corresponds to the distance between the symphysis pubis and the top of the uterus, in centimetres. At the beginning of the second trimester around 13 weeks of gestational age, the fundal height is around 13 cm. However, there is some variation in the fetal growth, and it is not unusual that some women present a fundal height that is slightly smaller or larger than expected. In addition, quickening (i.e. the first detection by the woman of fetal movement) is used in many countries to determine if a woman is in her second trimester. However, it is not a marker of the beginning of the

1 “Moderate transmission” areas are meso-endemic areas in which the prevalence rate of malaria is 11–50% during most time of the year among children from 2 to 9 years old. In these areas, the maximum prevalence of malaria infection occurs in childhood and adolescence, though it may not be unusual to acquire the first infection as an adult. “High transmission” areas are hyperendemic and holo-endemic areas in which the prevalence rate of malaria is over 50% during most time of the year among children from 2 to 9 years old. In these areas, practically all individuals have acquired their first infection by late infancy or early childhood. (6)

- 2 - second trimester. While some pregnant women experience quickening as early as 16 weeks, others may not do so until 20 weeks of gestation.

WHO 2013/2014 policy recommends that SP should be given at each scheduled ANC visit except during the first trimester, and it can be repeated every month with the doses given at least one month apart until the time of delivery. The previous WHO policy recommendation proposed that IPTp-SP be delivered at each ANC visit in order to ensure that pregnant women received at least two doses of SP. However, this resulted in many countries adopting a policy that recommended the administration of SP only twice during pregnancy. The new WHO policy recommendation calls for the administration of IPTp-SP at each ANC visit, starting as early as possible during the second trimester. This recommendation reflects the need to increase the number of SP doses. This decision was based on the most recent evidence that among pregnant women in sub-Saharan Africa, intermittent preventive treatment in pregnancy with 3+ doses of SP was associated with a higher birth weight and lower risk of LBW than compared to the standard two-dose regimens (7). The new policy does not refer to a specific number of doses, as experience has shown that once the policy states a specific number of doses, even if qualified (e.g. “minimum of three doses,” “three or more doses,” or “at least three doses”), this becomes a programmatic target for many countries. The new policy, calling only for administration of IPTp-SP at each ANC visit except during the first trimester and with the doses given at least one month apart until the time of delivery, is not restrictive. While the new policy does not recommend a maximum number of doses of IPTp-SP, as previously noted, SP can be safely administered from the beginning of the second trimester until delivery, provided that doses are given one month apart. The last dose of SP can be administered up to the time of delivery without safety concerns. Previously there was concern that the administration of SP late in pregnancy could result in kernicterus. However, review of the extensive evidence on the use of SP and related compounds to prevent maternal malaria and treat congenital toxoplasmosis in near-term pregnant women and newborns demonstrated no clinical association between SP use and kernicterus.

If a woman presents to an antenatal care clinic with symptoms of malaria, these symptoms should be investigated before the administration of IPTp-SP. If the woman tests positive for malaria – by either microscopy or rapid diagnostic test (RDT) – she should be treated following national case management guidelines. If she is negative, she should receive IPTp-SP.

Co-administration of other medication. High doses of folic acid (i.e. daily dose equal to 5 mg or above) have been shown to counteract the efficacy of SP as an antimalarial, and thus only the low dose (i.e. 0.4 mg daily) should be co-administered with SP.

. Folic acid requirements are increased in pregnancy because of the rapidly dividing cells in the fetus and elevated urinary losses. WHO recommends iron and folic acid supplementation in pregnant women at a dose of 30-60 mg of elemental iron6 plus 0.4 mg of folic acid, daily (8). Every effort should be made to ensure that low dose folic acid (0.4 mg or 400 micrograms) is available and provided as part of routine antenatal care. . If a woman is diagnosed with anaemia in a clinical setting, WHO recommends treatment with 120 mg elemental iron daily (given in two separate doses, i.e., 60 mg in the morning and 60 mg in the evening) and 0.4 mg folic acid supplementation until her haemoglobin concentration rises to normal (18). She can then switch to the standard antenatal dose to prevent recurrence of anaemia, i.e. 30-60 mg of elemental iron plus 0.4 mg of folic acid, daily. . Folic acid at a dose of 5 mg is recommended for prevention of neural tube defects among women who have previously delivered a child with a neural tube defect. Folic acid supplementation after the first month of pregnancy will not prevent neural tube defects, as the neural tube closes by day 28 of pregnancy. Folic acid at a daily dose equal or above to 5 mg should not be given together with SP as this counteracts the antimalarial efficacy of SP. There is presently no scientific consensus on how long SP should be withheld. However, expert opinion states that withholding SP for two weeks after administration of 5 mg or more of folic acid, as is the practice in many countries, is likely to be too short an interval between treatments. Strong advice should be given by the health care provider to the pregnant women to use her ITN and to immediately come back in case of malaria symptoms for proper diagnosis and treatment.

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Moreover, SP should not be administered concurrently with co-trimoxazole prophylaxis due to their redundant mechanisms of action and synergistic worsening of adverse drug reactions. Therefore, HIV-infected pregnant women who are already receiving co-trimoxazole prophylaxis should not receive IPTp-SP (4).

Monitoring. WHO recommends monitoring the implementation of the new IPTp-SP policy via routine monitoring systems and household surveys. Each dose of IPTp-SP given should be recorded in order that the proportion of the pregnant women receiving each dose (i.e., IPTp-1, IPTp-2, IPTp-3 and IPTp-4) can be monitored. Health providers should be encouraged to administer and record SP doses given at each scheduled antenatal visit. ANC registers and the Health Management Information System (HMIS) forms are being / will need to be adapted to now also record the third and fourth doses of SP for IPTp-SP. Demographic Health Surveys (DHS) reports and certain national survey instruments have and also will need to be updated to monitor the implementation of the new recommendations.

8. Information supporting the public health relevance.

Malaria is one of the leading causes of illness, death, and lost economic productivity globally. While there has been successful scale-up and use of critical commodities, malaria still resulted in over 219 million cases and more than 435,000 deaths in 2017; most of the deaths occurred in children under five years of age and pregnant women. (9)

In sub-Saharan Africa (SSA), over 30 million pregnant women are annually exposed to infection from malaria (10). Of these, an estimated 10,000 pregnant women and up to 200,000 newborns die from malaria in pregnancy (MiP), primarily due to the infection from Plasmodium falciparum (11). Furthermore, recent data indicate that up to 20% of stillbirths in SSA are attributable to MiP (12).

Since October 2012, WHO has recommended that IPTp be given to all pregnant women at each ANC visits, starting as early as possible in the second trimester (i.e. not during the first trimester). Each IPTp-SP dose should be given at least 1 month apart, with at least three doses during each pregnancy. The expected benefits of are as follows:

. IPTp-SP prevents the adverse consequences of malaria on maternal and fetal outcomes, such as placental infection, clinical malaria, maternal anaemia, fetal anaemia, low birth weight and neonatal mortality (13).

. IPTp-SP has recently been shown to be highly cost-effective for both prevention of maternal malaria and reduction of neonatal mortality in areas with moderate or high malaria transmission (14).

. Despite the spread of SP resistance, IPTp-SP continues to provide significant benefit, resulting in protection against both neonatal mortality (protective efficacy 18%) and low birth weight (21% reduction in LBW) under routine programme conditions (15).

To date, 39 African countries have adopted this policy. However, there is an unacceptably low proportion of eligible pregnant women receiving IPTp with quality-assured sulfadoxine-pyrimethamine (SP): only an estimated 22% of pregnant women received three doses of IPTp-SP in 2017 (9). It has been estimated that if all women with at least 3 ANC visits in Africa received IPTp-SP, that an additional 215,000 (128,000± 318,000 95% crI) LBW deliveries could be prevented (16).

9. Review of benefits: summary of evidence of comparative effectiveness.

GRADE assessment (1). In a systematic review of IPTp, seven trials involving direct comparison of two doses of SP with three or more doses monthly were evaluated. The trials were conducted in Burkina Faso, Kenya, Malawi, Mali and Zambia between 1996 and 2008. In comparison with two doses of SP, three or more doses:

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. increased the mean birth weight by about 56 g (95% CI, 29–83; seven trials, 2190 participants, high-quality evidence); . reduced the number of low-birth-weight infants by about 20% (RR, 0.80; 95% CI, 0.69–0.94; seven trials, 2190 participants, high-quality evidence); . reduced placental parasitaemia by about 50% (RR, 0.51; 95% CI, 0.38–0.68; six trials, 1436 participants, high-quality evidence); and . reduced maternal parasitaemia by about 33% (RR, 0.68; 95% CI, 0.52– 0.89; seven trials, 2096 participants, high-quality evidence).

The trials conducted to date have not been large enough to detect or exclude effects on spontaneous miscarriage, stillbirth or neonatal mortality (very low quality evidence).

The guideline development group noted that the beneficial effects were obvious in women in their first and second pregnancies. There was less information on women in their third or later pregnancy, but the available information was consistent with benefit. (17)

In the systematic review summarized above, the reduction in risk for low birth weight was consistent for a wide range of levels of resistance to SP. The group that received three or more doses also had less placental malaria. There were no differences in serious adverse events between the two groups. On the basis of these results, WHO now recommends that, in areas of moderate-to-high malaria transmission of Africa, IPTp-SP be given to all pregnant women at each scheduled antenatal care visit, starting as early as possible in the second trimester, provided that the doses of SP are given at least 1 month apart. The objective is to ensure that at least three doses are received.

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The relevant GRADE table as included in the third edition of the WHO Guidelines for the treatment of malaria are displayed below (MTG, Annex 4, A4.19)

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10. Review of harms and toxicity: summary of evidence of safety.

Despite the known side effects associated with sulfonamides, SP for intermittent preventive treatment in pregnancy is generally very well tolerated. Mild and transient side effects including nausea, vomiting, weakness and dizziness have been reported by some women, particularly with the first dose of SP. Studies have demonstrated that side effects tend to decrease with the administration of further doses (18, 19). Side effects should be discussed openly and managed in the ANC. (20)

To complement this information, below safety profile for SP is taken from the WHO Guidelines for the Treatment of malaria (3rd Edition) (1):

Adverse events. At the recommended doses, SP is generally well tolerated. The adverse effects reported are mainly those associated with sulfonamides, including gastrointestinal disturbances, headache, dizziness and skin reactions such as photosensitivity, rash, pruritus, urticaria and slight hair loss (21-25). Potentially fatal skin reactions, namely erythema multiforme, Stevens–Johnson syndrome and toxic epidermal necrolysis, may also occur (21). Leukopenia, thrombocytopenia, megaloblastic anaemia, haemolytic anaemia (probably related to G6PD deficiency), crystalluria, haematuria, oliguria and hepatitis have also been reported. There have been isolated case reports of serum sickness, allergic pericarditis and pulmonary infiltrates resembling eosinophilic or allergic alveolitis.

Contraindications. SP, alone or in combination with or , is contraindicated in:

. individuals with known hypersensitivity to pyrimethamine, sulfonamides and related compounds; . patients with documented megaloblastic anaemia due to folate deficiency; . premature or newborn infants in the first 2 months of life, because of the immaturity of their enzyme systems; and . HIV-infected patients receiving cotrimoxazole prophylaxis against opportunistic infections.

Caution. If skin eruptions, cytopenia or a bacterial or fungal super-infection occurs, use of SP should be discontinued. Caution is advised in repeated administration of SP to patients with blood dyscrasias and those with renal or hepatic failure, in whom the drugs accumulate.

Drug interactions. The following demonstrated and potential interactions have been observed:

. Demonstrated interactions: ‒ Reduced effectiveness with high doses >5mg of folic acid (26). ‒ Avoid concurrent or trimethoprim + due to increased risk for severe cutaneous reactions (27)

. Potential interactions: ‒ Additive haematological toxicity with myelo suppressants such as methotrexate, daunorubicin and cytarabine (28) ‒ Hepatotoxicity with lorazepam (29) ‒ Kaolin (30) may reduce absorption ‒ May displace , phenytoin and warfarin from plasma protein, possibly increasing toxicity (28)

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11. Summary of available data on comparative cost and cost-effectiveness of the medicine.

SP is an inexpensive medicine, and most countries already have a delivery system for IPTp-SP in place, which is often integrated into a comprehensive Focused Antenatal Care (FANC) package. Countries, however, may require investments associated with reviewing national guidelines to ensure harmonization and disseminating updated training packages and Information, Education and Communication (IEC) materials or job aids, as well as adapting tools to capture new indicators. (20) In comparison to placebo, in Mozambique, delivery of two doses of IPTp-SP has been estimated to cost $41.46 USD (CI 95% 20.50, 96.70) per maternal outpatient visit averted. This same study estimated an incremental cost effectiveness ratio (ICER) of $1.08 USD (CI 95% 0.43, 3.48) per DALY averted (31). Additionally, using data from seven countries, the incremental cost-effectiveness of IPTp3 compared to IPTp2 has been estimated at $7.28 USD (32).

In some countries of sub-Saharan Africa, transmission of malaria has been reduced substantially due to the successful implementation of malaria control efforts. At present there is insufficient evidence on the level of malaria transmission below which the risks and cost for IPTp-SP exceed its benefits and when it should be stopped. Because of natural fluctuations in the incidence of malaria from year to year, the low cost of IPTp-SP and the challenges of reintroducing IPTp after a potential withdrawal, countries should be cautious in discontinuing IPTp-SP until more information is obtained that would allow the formulation of more specific guidelines (7). The WHO recommendations on intermittent screening and treatment in pregnancy and the safety of ACTs in the first trimester state that (33):

. IPTp-SP remains highly cost-effective in preventing the adverse consequences of malaria on maternal and fetal outcomes, and should therefore be actively scaled up in line with the current WHO recommendations. IPTp-SP also remains effective in areas where quintuple- mutant haplotypes of Plasmodium falciparum to SP are highly prevalent. Further research on the relationship of SP resistance markers and IPTp effectiveness should be done, particularly in areas where transmission and thus maternal immunity have declined substantially in recent years.

. The threshold level of malaria transmission below which IPTp-SP is no longer cost-effective has not been identified. Therefore, in areas where IPTp-SP is implemented and transmission has been reduced to low levels as a result of successful control strategies, WHO recommends continued IPTp-SP implementation until the area approaches interruption of transmission.

. An association between sextuple mutant haplotypes of P. falciparum and decreased birth weight has been reported in observational studies in a few sites in East Africa. Further studies are required to assess this and to devise the best and most cost-effective prevention strategies in areas of very high SP resistance. One potential strategy to be tested is to provide a single RDT screening and ACT treatment at the first ANC visit during the second trimester, in addition to the continued delivery of IPTp-SP.

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Regulatory information

12. Summary of regulatory status and market availability of the medicine.

Sulfadoxine-pyrimethamine as fixed-dose combination tablet is currently available on the market from two quality-assessed sources: One product is approved by the Cyprus regulatory authorities, the other is WHO prequalified. Details on the corresponding products are available on the List of Malaria Pharmaceutical Products classified according to the Global Fund Quality Assurance Policy (accessible via the following link: https://www.theglobalfund.org/media/4756/psm_productsmalaria_list_en.pdf) and the list of WHO prequalified products for malaria (accessible via the following link: https://extranet.who.int/prequal/).

Only SP of proven quality should be used. In order to preserve SP efficacy for IPTp-SP, all possible efforts should be made to avoid SP use as monotherapy for the treatment of clinical cases of malaria. Reserving available SP stocks for use as intermittent preventive treatment in pregnancy at antenatal care clinics limits the risk of stock outs due to non-recommended use as monotherapy for clinical cases. Reserving SP for dispensing from the pharmacy and giving SP to take at home can both inhibit the use of IPTp-SP by pregnant women.

13. Availability of pharmacopoeial standards (British Pharmacopoeia, International Pharmacopoeia, United States Pharmacopoeia, European Pharmacopeia).

The international Pharmacopoeia, 7th Edition, 2017, states below monograph for SP (accessible via the following link: http://apps.who.int/phint/en/p/docf/anchor,get-started.html):

Monographs: Dosage forms: Specific monographs: Sulfadoxine and pyrimethamine tablets (Sulfadoxini et pyrimethamini compressi)

Category. Antimalarial.

Storage. Sulfadoxine and pyrimethamine tablets should be kept in a well-closed container, protected from light.

Additional information. Strength in the current WHO Model list of essential medicines: 500 mg sulfadoxine and 25 mg pyrimethamine.

Strength in the current WHO Model list of essential medicines for children: 500 mg sulfadoxine and 25 mg pyrimethamine.

Requirements

Comply with the monograph for "Tablets".

Definition. Sulfadoxine and pyrimethamine tablets contain Sulfadoxine and Pyrimethamine. They contain not less than 90.0% and not more than 110.0% of the amounts of sulfadoxine (C12H14N4O4S) and pyrimethamine (C12H13ClN4) stated on the label.

Identity tests

A. Carry out test A.1 or, where UV detection is not available, test A.2.

A.1 Carry out the test as described under 1.14.1 Thin-layer chromatography, using silica gel R6 as the coating substance and a mixture of 75 volumes of ethyl acetate R, 25 volumes of methanol R and 1 volume of glacial acetic acid R as the mobile phase. Apply separately to the plate 10 μl of each of the following two solutions in methanol R. For solution (A) shake a quantity of the powdered tablets containing about 100 mg of Sulfadoxine for 5 minutes with 20 mL, filter and use the filtrate. For solution (B) use 5 mg of sulfadoxine RS and 0.25 mg of pyrimethamine RS per mL. After removing the plate from the chromatographic chamber allow it to dry in a current of air and examine the chromatogram in ultraviolet light (254 nm).

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The two principal spots obtained with solution (A) correspond in position, appearance and intensity to those obtained with solution (B).

A.2 Carry out the test as described under 1.14.1 Thin-layer chromatography, using the conditions described above under test A.1 but using silica gel R5 as the coating substance. Dip the plate in modified Dragendorff reagent TS. Examine the chromatogram in daylight.

The two principal spots obtained with solution (A) correspond in position, appearance and intensity to those obtained with solution (B) (the spot due to pyrimethamine is faintly visible).

B.See the test described under Assay. The retention times of the two principal peaks in the chromatogram obtained with solution (1) are similar to those in the chromatogram obtained with solution (4).

Dissolution

Carry out the test as described under 5.5 Dissolution test for solid oral dosage forms , using as the dissolution medium, 1000 mL of hydrochloric acid (~4 g/l) TS and rotating the paddle at 75 revolutions per minute. At 30 minutes withdraw a sample of about 5 mL of the medium through an in-line filter and use the filtrate. Determine the content of sulfadoxine (C12H14N4O4S) and pyrimethamine (C12H13ClN4) in the filtrate according to the method as described under Assay and preparing solution (4) under Assay as follows: dilute 10.0 mL of solution (2) and 2.0 mL of solution (3) to 20.0 mL with hydrochloric acid (~4 g/l) TS.

For each of the six tablets calculate the total amount of sulfadoxine (C12H14N4O4S) and pyrimethamine (C12H13ClN4) in the medium from the results obtained. For both substances the amount in solution for each tablet is not less than 80% of the amount declared on the label. For either substance, if the amount obtained for one of the six tablets is less than 80%, repeat the test using a further six tablets; the average amount for all 12 tablets tested is not less than 75% and no tablet releases less than 60%.

Sulfadoxine-related substances

Carry out the test as described under 1.14.4 High-performance liquid chromatography, using a stainless steel column (25 cm x 4.6 mm) packed with particles of silica gel, the surface of which has been modified with chemically-bonded octadecylsilyl groups (5 μm).

As the mobile phase use a solution prepared as follows: dissolve 10 mL of glacial acetic acid R and 0.5 mL of triethylamine R in about 800 mL of water R, dilute to 1000 mL and adjust the pH to 4.2 by adding sodium hydroxide (~400 g/l) TS. Mix 850 mL of this solution with 120 mL of acetonitrile R and 30 mL of methanol R.

For solution (1) weigh and powder 20 tablets. Transfer a quantity of the powder containing about 200 mg of Sulfadoxine into a 100 mL volumetric flask. Add 35 mL of acetonitrile R and sonicate for about 10 minutes. Allow to cool to room temperature and make up to volume with mobile phase. Filter a portion of this solution through a 0.45 μm filter, discarding the first few mL of the filtrate. For solution (2) dilute 1 mL of solution (1) to 200 mL with the mobile phase.

For solution (3) prepare a solution containing about 1 mg of sulfadoxine RS and about 0.5 mg of sulfamethoxazole R per mL in acetonitrile R. Dilute 10 mL of this solution to 100 mL with the mobile phase.

Operate with a flow rate of 2 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 270 nm.

Inject separately 100 μl each of solutions (1), (2) and (3). Record the chromatograms for about 3.5 times the retention time of sulfadoxine (to ensure that pyrimethamine is eluted).

In the chromatogram obtained with solution (1) the following impurity peaks, if present, are eluted at the following relative retention with reference to sulfadoxine (retention time about 18 minutes): impurity A () about 0.1; impurity B about 0.2; impurity D about 0.3; impurity C about 1.4. A peak due to pyrimethamine has a relative retention of about 2.7. The test is not valid unless in the chromatogram obtained with solution (3) the resolution between the peaks due to sulfadoxine and to sulfamethoxazole (with relative retention of about 1.1 with reference to sulfadoxine) is at least 2.

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In the chromatogram obtained with solution (1) the area of any peak, other than the peaks due to sulfadoxine and to pyrimethamine, is not greater than the area of the peak due to sulfadoxine in the chromatogram obtained with solution (2) (0.5%). The sum of the areas of all peaks, other than the peaks due to sulfadoxine and pyrimethamine, is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with solution (2) (0.05%).

Assay

• Either method A or B may be applied.

A. Carry out the test as described under 1.14.4 High-performance liquid chromatography, using a stainless steel column (25 cm x 4.6 mm) packed with particles of silica gel, the surface of which has been modified with chemically-bonded octadecylsilyl groups (5 μm).

As the mobile phase use a solution prepared as follows: dissolve 10 mL of glacial acetic acid R and 0.5 mL of triethylamine R in about 800 mL of water R, dilute to 1000 mL and adjust the pH to 4.2 by adding sodium hydroxide (~400 g/l) TS. Mix 800 mL of this solution with 200 mL of acetonitrile R.

For solution (1) weigh and powder 20 tablets and transfer a quantity of the powder containing about 0.50 g of Sulfadoxine, accurately weighed, into a 200 mL volumetric flask. Add about 70 mL of acetonitrile R and sonicate for 10 minutes. Allow to cool to room temperature, make up to volume using the mobile phase and sonicate for 10 minutes. Dilute 5 mL of this solution to 25 mL with the mobile phase and filter a portion of this solution through a 0.45 μm filter, discarding the first few mL of the filtered solution. For solution (2) transfer 25 mg of sulfadoxine RS, accurately weighed, to about 10 mL of acetonitrile R, sonicate until dissolved and dilute to 25.0 mL with the mobile phase. For solution (3) transfer 25 mg of pyrimethamine RS, accurately weighed, to about 35 mL of acetonitrile R, sonicate until dissolved and dilute to 100.0 mL with the mobile phase. For solution (4) dilute 10.0 mL of solution (2) and 2.0 mL of solution (3) to 20.0 mL with the mobile phase.

Operate with a flow rate of 2 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 227 nm.

Inject 20 µl of solution (4). The assay is not valid unless the resolution between the peaks due to sulfadoxine and to pyrimethamine, eluting in this order, is at least 5. The run time for the analyses is not less than 25 minutes.

Inject separately 20 µl each of solutions (1) and (4).

Measure the areas of the peak responses obtained in the chromatograms from solutions (1) and (4) and calculate the content of sulfadoxine (C12H14N4O4S) and pyrimethamine (C12H13ClN4) in the tablets.

B. Use the average of 10 individual results obtained for each of sulfadoxine and pyrimethamine in the test for uniformity of content.

Uniformity of content

The tablets comply with the test for 5.1 Uniformity of content for single-dose preparations, using the following method of analysis.

Carry out the test as described under 1.14.4 High-performance liquid chromatography, using the chromatographic conditions as described under Assay, Method A.

For solution (1) transfer one powdered tablet to a 200 mL volumetric flask. Add about 70 mL of acetonitrile R and sonicate for 10 minutes. Allow to cool to room temperature, make up to volume using the mobile phase and sonicate for 10 minutes. Dilute 5 mL of this solution to 25 mL with the mobile phase and filter a portion of this solution through a 0.45 μm filter, discarding the first few mL of the filtered solution. For solution (2) transfer 25 mg of sulfadoxine RS, accurately weighed, to about 10 mL of acetonitrile R, sonicate until dissolved and dilute to 25.0 mL with the mobile phase. For solution (3) transfer 25 mg of pyrimethamine RS, accurately weighed, to about 35 mL of acetonitrile R, sonicate until dissolved and dilute to 100.0 mL with the mobile phase. For solution (4) dilute 10.0 mL of solution (2) and 2.0 mL of solution (3) to 20.0 mL with the mobile phase.

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Operate with a flow rate of 2 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 227 nm.

Inject 20 µl of solution (4). The assay is not valid unless the resolution between the peaks due to sulfadoxine and to pyrimethamine, eluting in this order, is at least 5. The run time for the analyses is not less than 25 minutes.

Inject separately 20 µl each of solution (1) and (4).

Measure the areas of the peak responses obtained in the chromatograms for solutions (1) and (4) and calculate the content of sulfadoxine (C12H14N4O4S) and pyrimethamine (C12H13ClN4) in each tablet.

Impurities

A. 4-aminobenzenesulfonamide (sulfanilamide),

B. 4-amino-N-(6-hydroxy-5-methoxypyrimidin-4-yl)benzenesulfonamide,

C.N-{4-[(5,6-dimethoxypyrimidin-4-yl)sulfamonyl]phenyl}acetamide,

D. 5,6-dimethoxypyrimidin-4-amine.

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