Title: Method development and validation for Accurate quantification OF PHARMACEUTICAL Drugs and its Impurities using uplc and HPLC as a Separation Tool
Literature survey of Drugs under study
- General and Physical
Amoxicillin is a semi synthetic amino penicillin antibiotic with bactericidal activity. The chemical name of Amoxicillin is “(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-abicyclo [3.2.0] heptane-2-carboxylic acid”. The molecular weight is 365.4 g/mol, it is slightly soluble in water.
Figure 4.1 Chemical structure of Amoxicillin as Trihydrate.
- Mechanism of action:
Amoxicillin trihydrate is the anhydrous form of a broad-spectrum, semisynthetic aminopenicillin antibiotic with bactericidal activity. Amoxicillin binds to and inactivates penicillin-binding proteins (PBPs) positioned on the inner membrane of the bacterial cell wall. Inactivation of PBPs interferes with the cross-linkage of peptidoglycan chains essential for bacterial cell wall strength and rigidity. This interrupts bacterial cell wall synthesis and results in the weakening of the bacterial cell wall and causes cell lysis. So, Amoxicillin is useful only actively growing and cell wall synthesizing bacteria.
- Dosage, Administration and pharmacology [105, 154, 155]
Amoxicillin orally administered in different strengths and dosage forms i.e. as Capsules, Tablets, Oral suspensions and injections. For adults that are older than three months the treatment should be sustained for at least 48 to 72 hours beyond the time that the patient becomes asymptomatic of bacterial suppression has been obtained. It is suggested that no less than 10 days treatment for any kind of infection caused by Streptococcus pyogenes bacteria to prevent the occurrence of severe rheumatic fever. In a few infections, therapy possibly required for several weeks. It may be needed to continue clinical follow-up for more than a few months after termination of therapy. Dosing in Neonates and Infants aged below 12 Weeks, treatment should be given for at least 48 to 72 hours beyond the time that the patient becomes asymptomatic of bacterial suppression has been obtained. Because of partly developed renal function affecting removal of Amoxicillin in this age group, the optional higher dose of Amoxicillin is 30 mg/kg/day which is divided every 12 hours, currently there are no dosing recommendations for pediatric patients with impaired renal function.
In dosing for H. pylori Infection, triple therapy, the recommended Amoxicillin oral dose for adult is 1 gram twice a day for 14 days. In duel therapy it is needed three times a day for 14 days.
Patients with impaired renal function normally do not need a dose reduction unless the impairment is severe. The dosages are differed for severely impaired patients based on a glomerular filtration rate. Patients should not receive the 875-mg dose with glomerular filtration rate of less than 30 mL/min, 10 to 30 mL/min patients should receive 500 mg or 250 mg in every 12 hours intervel, depending on the severity level of the infection, less than 10 mL/min should receive 500 mg or 250 mg every 24 hours, also it depends on severity of the infection.
Based on severity of the infection, Hemodialysis patients should receive 500 mg or 250 mg in every 24 hours, they should receive an additional dose both during and at the end of dialysis.
In pharmacology, the absorption of Amoxicillin is kind of stable within the presence of gastric acid and after oral administration it is absorbed rapidly. The food effect on the absorption of Amoxicillin from the tablets and suspension dosages has been partially investigated. Mean Amoxicillin pharmacokinetic parameters in 27 adult subjects from an open, two-part, single-dose crossover bioequivalence study in Fed state comparing 875 mg of Amoxicillin with 875 mg of Amoxicillin and Clavulanate potassium showed that the 875 mg tablet of Amoxicillin produces an AUC0-∞ of 35.4 ± 8.1 µg/hr/mL and a Cmax of 13.8 ± 4.1 µg/mL.
Amoxicillin diffuses readily into most of the body tissues and fluids, with the exception of brain and spinal fluid, except when meninges are inflamed. In blood serum, approximately 20% protein-bound is Amoxicillin. Following a one gram dose and by utilizing a special skin window technique to estimate levels of the antibiotic, it had been noted that therapeutic concentration levels were found in the interstitial fluid.
Metabolism and Excretion
The half-life of Amoxicillin is about one hour. Approximately 60% of an orally administered dose of Amoxicillin is excreted in the urine within 6 to 8 hours. After an orally administered dose of Amoxicillin the detectable serum levels are observed up to 8 hours. Since most of the Amoxicillin is excreted unchanged in the urine, its excretion is often delayed by simultaneous administration of probenecid.
- General and Physical:
Ampicillin is a semi synthetic amino penicillin antibiotic with bactericidal activity. The chemical name of Ampicillin is “(2S, 5R, 6R)-6-[[(2R)-2-amino-2-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid”. It is available in trihydrate form. The molecular formula as trihydrate is C16H19N3O4.3H2O, molecular weight is 403.45 g/mol, it is soluble in water, the melting range is between 202-208ºC.
Figure 4.2 Chemical structure of Ampicillin as Trihydrate.
- Mechanism of action:
Ampicillin (AMP) is semis-synthetic, beta lactam penicillin antibiotic with bactericidal activity. AMP binds and inactivates penicillin binding proteins (PBP) situated on the inner membrane of the bacterial cell wall (BCW). Inactivation of PBPs interferes with the chemical bond of peptidoglycan chains necessary for BCW strength and firmness. This disturbs the BCW synthesis and results in the weakening of the BCW and causes cell lysis. Ampicillin is very stable against hydrolysis by a variety of beta-lactamases, therefore, is employed in wide range of gram-positive and -negative infections.
- Dosage, Administration and pharmacology[105, 154, 155]
Ampicillin as Trihydrate is administered orally in different of strengths as Capsules, and Oral suspensions, Ampicillin as Sodium administered as Injection. Ampicillin capsules 500mg are administered in equally spaced doses for adults and children weighing over 20 Kg, for genitourinary or gastrointestinal tract infections except gonorrhea. Larger doses might need for severe or chronic infections. It is recommended in treatment of gonorrhea for both male and female adults, a single dose of 3.5 grams of Ampicillin is employed simultaneously with 1.0 gram of Probenecid. Physicians are cautioned to employ not less than the above recommended dosage for the treatment of gonorrhea. Follow-up cultures should be obtained from the same site(s) of infection seven to fourteen days after therapy. In female adults, it is also recommended to obtain culture test-of-cure from each the endocervical and anal canals. Extended intensive medical care is needed for complications like as prostatitis and epididymitis.
For respiratory tract infections in pediatric patients weighing less than 20 Kg, the recommended usual dose is 250 mg in equally spaced doses. For genitourinary or gastrointestinal tract infections, the recommended standard dose is 100 mg/kg/day total, for respiratory tract infections the standard dose is 50 mg/kg/day total, in equally divided and spaced doses three to four times daily. The recommended doses should not exceed for children and adults.
Ampicillin is germicidal at small concentrations and is clinically effective not solely against the gram-positive organisms typically prone to penicillin G, however conjointly against a range of gram-negative organisms. It is observed in the presence of gastric acid it is stable, also well absorbed from the gastrointestinal tract. It diffuses easily into most of body tissues and fluids, but, penetration into the cerebrospinal fluid and brain happens particularly with meningeal inflammation. Ampicillin is excreted for the most part unchanged in the urine and its excretion is delayed by simultaneous administration of probenecid which inhibits the renal tubular secretion of Ampicillin. In blood serum, Ampicillin is the least bound of all the penicillins, approximately a mean of 20 percent of the drug is bound to the plasma proteins as compared to 60 – 90% of the other penicillins. The administration of Ampicillin trihydrate capsules 500 mg dose fallout in a mean peak blood serum level of approximately 3.0 mcg/mL, the mean peak serum level of Ampicillin trihydrate for oral suspension for a 250 mg dose is nearly 2.3 mcg/mL.
- Penicillin V Potassium:
- General and Physical
Penicillin V Potassium is a semi synthetic amino penicillin antibiotic with bactericidal activity. The chemical name of Penicillin V Potassium is potassium; (2S,5R,6R) – 3, 3 – dimethyl – 7 – oxo – 6 – [(2-phenoxyacetyl) amino] – 4 – thia – 1 zabicyclo [3.2.0] heptanes – 2 – carboxylate”. The molecular formula as trihydrate is C16H17KN2O5S, molecular weight is 388.48 g/mol, it is soluble in water, the melting range is between 387-396 ºC.
Figure 4.3 Chemical structure of Penicillin V Potassium.
- Mechanism of action
Penicillin V potassium is the salt form of penicillin V, which is a member of the beta lactum antibiotic family with broad spectrum of germicidal activity. Penicillin V binds to and deactivates penicillin-binding proteins (PBPs) enzymes situated on the inner membrane of the bacterial cell wall (BCW), ensuing in the deteriorating of the BCW and cell lysis. PBPs take part in the terminal stages of assembling the BCW, and in restructuring the cell wall while it is under cell division. Inactivation of PBPs interferes with the bond of peptidoglycan chains necessary for BCW strength and inflexibility.
- Dosage, Administration and pharmacology [105, 154, 155]
Penicillin V Potassium is administered orally in different strengths as Tablets, and Oral solutions. The dosage of penicillin V should be determined based on sensitivity of the causative microorganisms and the severity of infection, and adjusted to the medical response of the patient. Based on type of infections, the usual dosage for adults and children 12 years and over are given as following;
Streptococcal infections: The recommended dose for mild to moderately severe upper respiratory tract and including scarlet fever and erysipelas is 125 to 250 mg (200,000 to 400,000 units) for every 6 to 8 hours for 10 days.
Pneumococcal infections; The recommended dose for mild to moderately severe respiratory tract, including otitis media is 250 to 500 mg (400,000 to 800,000 units) for every 6 hours until the patient has been a febrile for minimum 2 days.
Staphylococcal infections: The culture and sensitive tests should be performed for mild infections of skin and soft tissue. The recommended dose is 250 to 500 mg (400,000 to 800,000 units) in every 6 to 8 hours for 10 days.
Fusospirochetosis (Vincents infection) : The recommended dose for mild to moderately severe infections of the oropharynx is 250 to 500 mg (400,000 to 800,000 units) every 6 to 8 hours for 10 days.
For the prevention of recurrence rheumatic fever and/or chorea, 125 to 250 mg (200,000 to 400,000 units) dose is recommended twice daily on a ongoing basis. Usually a 2 gram of penicillin V (1 gram for children under 60 lbs.) is recommended before one hour of the procedure and one gram (500 mg for children under 60 lbs.) six hours later for prophylaxis against bacterial endocarditis in patients with congenital heart disease or rheumatic or other acquired valvular heart disease when undergoing dental procedures or surgical procedures of the upper respiratory tract.
Penicillin V gives an antiseptic action against penicillin sensitive microorganisms during the stage of active multiplication. It acts through the inhibition of bio-synthesis of mucopeptide cell wall. It is not active against the penicillinase producing bacterium, that embody many strains of staphylococci. The drug exerts high in vitro activity against staphylococci (except penicillinase producing strains), streptococci (groups A, C, G, H, L and M), and pneumococci. Different organisms, sensitive in vitro to penicillin V are Corynebacteriumdiphtheriae, Bacillusanthracis, Clostridia, Actinomycesbovis, Streptobacillusmoniliformis, Listeria monocytogenes, Leptospira, and Neisseria gonorrhoeae. Treponemapallidum is very sensitive.
The Penicillin V potassium salt (PVK) has the distinct advantage over Phenoxymethyl penicillin G (Penicillin G) in resistance to inactivation by gastric acid. Based on pharmacokinetics the mean blood levels measured are 2-5 times beyond the degree following an equivalent dose of oral penicillin G and additionally show much less individual subject variation. Once absorbed, approximately 80% is bound to serum protein. Tissue levels square measure highest in the kidneys and lowest in the liver, skin, and intestines. Low amounts are observed in all other body tissues and the cerebrospinal fluid.
Blood levels measured slightly higher when the Penicillin V is given on an empty stomach, however, it may be administered with food. The drug is excreted as fast as it is absorbed in patients with normal kidney function, however the recovery of the PVK from the urine indicates that only about 25% of the dose given is absorbed. Excretion is significantly delayed in neonates, young infants, and individuals with impaired kidney function.
- Clavulanate Potassium:
- General and Physical
Clavulanate Potassium is a salt form of Clavulanic acid. The acid is suicide inhibitor of bacterial beta-lactamase enzymes from streptomyces clavuligerus. The chemical name of Clavulanate potassium “potassium (2R,3Z,5R) – 3 – (2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo [3.2.0] heptane-2-carboxylate”. The molecular formula as trihydrate is C8H8KNO5, molecular weight is 237.25 g/mol, it is soluble in water.
Figure 4.4 Chemical structure of Clavulanate Potassium
- Mechanism of action
Clavulanate Potassium (CP) found as semi-synthetic beta lactamase inhibitor isolated from streptomyces. It contains a beta lactam ring and which binds strongly to beta lactamase at or near its active site; this protects other beta-lactam antibiotics from beta lactamase catalysis. This drug is administered along with beta lactamase prone penicillins to treat infections caused by beta lactamase producing organisms.
- Dosage, Administration and pharmacology [105, 154, 155]
Clavulanate Potassium is administered orally in combination of penicillins like Amoxicillin or Ticarcillin in different strengths such as Tablets, and Oral solutions. The usual Oral solutions of Amoxicillin / Clavulanate potassium per 5mL are ; 600 mg/42.9 mg, 200/28.5 mg , 400 /57 mg, 125 /31.25 mg, 250/62.5 mg,; tablets are 250/125mg, 500/125 mg 875 mg/125 mg. One 500mg tablet of Amoxicillin and Clavulanic acid (‘Augmentin’ is Brand product) administered every 12 hours or one 250-mg tablet of Augmentin every 8 hours. For severe respiratory tract infections, the recommended Augmentin dose is one 875-mg/125mg tablet for every 12 hours or one 500-mg tablet of for every 8 hours. Patients who have difficulty swallowing may be given the 125 or 250mg per 5 mL suspension in place of the 500/125mg tablet. The 200 mg/28.5mg or 400 mg/57mg per 5mL suspension may be administered in place of the 875mg/125mg tablet.
Since both the 250mg/125mg and 500mg/125mg tablets of Augmentin contain the same amount of Clavulanic acid substitution of 250mg tablets of Augmentin in place of 500mg is not allowed.
The 250mg tablet of Augmentin and the 250mg chewable tablet do not contain the same amount of clavulanic acid (as the potassium salt), hence they should not have substituted for each other, as they are not interchangeable. The 250mg tablet of Augmentin contains 125 mg of clavulanic acid, whereas the 250mg chewable tablet contains only 62.5 mg.
Based on the Amoxicillin drug substance, neonates and infants aged less than 12 Weeks (less than 3 Months), the recommended dose of Augmentin is 30 mg/kg/day divided every 12 hours.
- Literature survey of simultaneous estimation analytical methods for Amoxicillin, Ampicillin, Penicillin V potassium and Clavulanic acid
Amoxicillin: In literature survey, for Amoxicillin estimation alone by HPLC[86, 91, 106, 107, 111, 112,113]and by UV spectrophotometer were found. Among those methods the RP-HPLC method developed by Manzoor Ahamed et.al for the estimation of Amoxicillin in bulk and pharmaceutical formulations, Amoxicillin was chromatographed on a hypersil C18 column (250×4.6mm I.D., particle size 5μm) in a mobile phase consisting of potassium dihydrogen phosphate and methanol in the ratio 95:05 v/v. The mobile phase at a flow rate of 1.0 ml/min was pumped with detection at 283 nm. The detector response was linear in the concentration of 20-100μg/ml. The method precision was found to be less than 2%. The mean recovery of the drug from the solution was 99.39%.
Another HPLC method reported from few other methods is developed by M C Hsu and P W Hsu91. It is a reversed-phase liquid chromatographic method was developed for the assay of amoxicillin and its preparations. The methods linear calibration range was 0.2 to 2.0 mg/ml (r = 0.9998), and drug recoveries were greater than 99%. The HPLC assay results were compared with those obtained from a microbiological assay of bulk drug substance and capsule, injection, and granule formulations containing amoxicillin and degraded amoxicillin. At the 99% confidence interval level, there was no significant inter method differences were noted for the paired results. Commercial formulations also analyzed and the results obtained by the proposed method were comparable with those found by the microbiological method. The results indicated that the proposed method is a suitable substitute for the microbiological method for assays and stability studies of amoxicillin preparations.
Ampicillin: In literature survey, few methods were found for Ampicillin, it is estimated alone by UV spectrophotometer , by HPLC [111,112] and in combination  by HPLC. Wenhong Luo  et.al, was developed one RP-HPLC method using fluorescence detection was developed for the simultaneous determination of amoxicillin and Ampicillin residues in raw and processed bovine milk. Aliquots of raw or processed milk (5 mL) were diluted to 40 mL with 0.01 M KH2PO4 (pH 4.5) buffer, and the soluble proteins were precipitated with the addition of sodium tungstate and sulfuric acid followed by centrifugation. The drug residues were concentrated by passing the supernatant through a C18 solid phase extraction cartridge. Amoxicillin and Ampicillin were eluted from the cartridge and reacted with salicylaldehyde to form fluorescent derivatives, which were then analyzed with liquid chromatography and fluorescence detection. Average recoveries of spiked amoxicillin and Ampicillin at 5, 10, and 20 ng/mL were >80%, with coefficients of variation (CV) <5%. The limit of detection (LOD) and limit of quantitation (LOQ) for Amoxicillin were 1.1 and 2.4 ng/mL, respectively. The LOD and LOQ for Ampicillin were 1.0 and 1.7 ng/mL, respectively.
One of the few methods found is the one developed by Ashnagar A and Gharib Naseri.N 92. As per this the purity of the standard active ingredients of the various dosage forms of three penicillins i.e., Amoxicillin, Cloxacillin and Ampicillin were investigated and determined by HPLC technique. The analyses were made by using a Knauer (Germany) Spherimage-80, ODS, 2-5 µm C18 column with 30 cm length, and i.d. 4.5 mm. A 20 µL injection volume is used. Each individual sample and the standard solutions were introduced separately onto the reverse phase column of an HPLC instrument which was equipped with ECW 2000 software of Knauer, Germany. The results obtained in this research have shown that the purity percentage of the active ingredients of the standard powder and the various dosage forms of all the drugs used, were 100%.
Penicillin V Potassium: In extensive literature survey for Penicillin v Potassium resulted few methods alone by HPLC [88, 93, 111, 112]. Among those, one RP-HPLC method developed by Baghel U.S et.al  for determination of Phenoxymethy penicillin potassium from tablet formulations. The determination was carried out on a Hypersil, ODS, C-18 (30cm*4mm) 5µ column using a mobile phase of water: acetonitrile: glacial acetic acid (500:500:5.75). The flow rate and run time were 1ml/min and 10 min respectively. The method was reproducible with theoretical plates and % RSD, but, the sensitivity of the method is low.
One of the few methods reported was by M.H. Sorouraddin, et.al  as per this a simple and selective method for penicillin V potassium (PVK) determination by chemiluminescence (CL) was developed. Oxidation of PVK by alkaline hydrogen peroxide produces CL, which is greatly enhanced by N, N-dimethyl formamide (DMF) and N-cetyl N, N, N-trimethylammonium bromide (CTMAB). Optimum conditions were established using luminometry. There is a linear relationship between the chemiluminescent peak height and the amount of PVK within the range 0.5 – 129.5 mg L-1, with a detection limit of 0.2 mg L-1. The coefficient of variation was 1.2% for 40 mg L-1 PVK solution (n = 7). The method is very simple, has high sensitivity and good selectivity, and is usable for process control. It was successfully utilized for the determination of PVK in pharmaceuticals and spiked human urine.
Clavulanic acid: In literature survey, few methods were resulted for Clavulanate Potassium estimation individually by HPLC [89, 94, 111, 112]. The one RP-HPLC method  developed by Durga et.al., for determination of simultaneous estimation of Clavulanic acid in combination of Amoxicillin from injectable dosage form. The separation was done using a mobile phase composition consists ratio of 95:5 (v/v) of pH 5.0 buffer and Methanol on Inertsil C18 column (250 × 4.0 mm, 4 μm) with detector wavelength at 220 nm at a flow rate of 1 ml/minute. The photodiode array detector (PDA) was used in stress studies detection. The order of elution of peaks was Clavulanic acid followed by Amoxicillin. The method’s linear calibration range was between 79.51 – 315.32 μg/mL for Amoxicillin and 17.82 – 67.90 μg/mL for Clavulanic acid. The method was proven stability indicating by subjecting the drugs to stress conditions as per ICH recommended test conditions such as alkaline and acid hydrolysis, oxidation, photolysis, thermal degradation and resolution of the degradation products found satisfactory. The sample solution stability studies reveal that Amoxicillin and Clavulanic acid were found to be stable up to 24 hours. The method validation data showed satisfactory results for precision, linearity, specificity, limit of detection, limit of quantification and robustness.
One of the few methods reported was developed by S.S. Jadhav et.al94 is a RP-HPLC method involving UV detection was developed and validated for determination and quantification of Amoxycillin Trihydrate and Potassium Clavulanate. Chromatography was carried out on pre-packed Inertsil C18 (5µm, 250×4.6mm) column using filtered and degassed mixture of Methanol: Aqueous disodium hydrogen phosphate pH 5 as mobile phase at flow rate of 1.0 ml/min and effluent was monitored at 249 nm. The method was validated in terms of Linearity, precision, accuracy, specificity, limit of detection, limit of quantification as per the ICH guidelines. The assay was linear over the concentration range of Amoxycillin trihydrate and potassium Clavulanate respectively. The accuracy and precision were found to be within the specified limits. The method does require only 9 minutes as run time for analysis which proves its adoptability.
The above literature survey for Amoxicillin, Ampicillin, Penicillin V Potassium and Clavulanic acid resulted that there was no single and sensitive method to estimate all four compounds simultaneously at lower concentration of 5 µg/mL.
- General and Physical
Acetaminophen is an Analgesic antipyretic derivative of acetanilide. Acetaminophen possesses weak anti-inflammatory properties and is used as a common analgesic. The chemical name of Acetaminophen is “N-(4-hydroxyphenyl) acetamide”. The molecular formula as trihydrate is C8H9NO2, molecular weight is 151.16 g/mol, it is soluble in water, the melting range is between 168ºC.
Figure 4.6 Chemical structure of Acetaminophen
- Mechanism of action :
Acetaminophen is also called as Paracetamol and is an extensively used analgesic and antipyretic drug. It is used for the relief of fever, headaches, and other minor aches and pains. It is a major API in number of cold and flu medications and many prescription analgesics. It is extremely safe in standard doses, due to its wide availability, deliberate or accidental overdoses are commonly found. Unlike other common analgesics such as Aspirin and Ibuprofen, it has no anti-inflammatory properties or effects on platelet function. It is not a member of the class of drugs known as non-steroidal anti-inflammatory drugs or NSAIDs. Unlike NSAIDS at therapeutic doses it does not infuriate the lining of the abdomen nor affect blood coagulation, kidney function, or the fetal ductus arteriosus. Like NSAIDs and unlike opioid analgesics, acetaminophen does not cause euphoria or change mood in any way. Acetaminophen and NSAIDs have the advantage of being fully free from issues with addiction, dependence, tolerance and withdrawal. Acetaminophen is administered on its own or in with combination Pseudoephedrine, Dextromethorphan, Chlorpheniramine, Guaifenesin, Diphenhydramine, Doxylamine, Codeine, Hydrocodone, or oxycodone.
Acetaminophen is a p-aminophenol derivative including analgesic and antipyretic behavior. Although the exact mechanism through which acetaminophen exert its effects has yet to be fully determined. It may inhibit the nitric oxide (NO) pathway mediated by a various neurotransmitter receptors as well as N-methyl-D-aspartate (NMDA) and substance P, resulting in elevation of the pain threshold. The antipyretic activity could result from inhibition of prostaglandin synthesis and discharge in the central nervous system (CNS) and prostaglandin-mediated effects on the heat regulating center in the anterior hypothalamus.
- Dosage, Administration and pharmacology [105, 154, 155, 156]
Acetaminophen is administered orally in different strengths as Tablets and Oral solutions. It is administered in combination such as Phenylephirine HCl, Guaifenesin, Pseudoephedrine, Dextromethorphan HBr, Chlorpheniramine, Diphenhydramine, Doxylamine, codeine, Hydrocodone, or Oxycodone. The maximum daily dose is based on all routes of administration and all products containing Acetaminophen.
Maximum daily dose(MDD) and dosing recommendations might differ by product, some manufacturers have decreased the MDD to protect consumers from accidental overdoses, the following are the common dosages for adults in treatment of fever and pain,
Body weight 50 kg or greater: The usual dose is 1000 mg for every 6 hours or 650 mg for every 4 hours, the maximum Single Dose should be 1000 mg and the minimum dosing interval should be every 4 hours.
Body weight less than 50 kg: The usual dose is 15 mg/kg for every 6 hours or 12.5 mg/kg for every 4 hours, the maximum single dose should be 15 mg/kg.
Pediatric Dosage: The Usual pediatric dosages for pain and fever for Age 2 to 12 years is 15 mg/kg or 12.5 mg/kg for every 4 hours, the maximum Single Dose should be 15 mg/kg and not to exceed 750 mg.
Acetaminophen features a spectrum of action like that of NSAIDs and resembles mainly the COX-2 selective inhibitors. Acetaminophen is, on average, a weaker analgesic than NSAIDs or COX-2 selective inhibitors however it is most commonly preferred because of its higher tolerance. Despite the similarities to NSAIDs, the mode of action of Acetaminophen has been unsure, however it is currently usually accepted that it inhibits COX-1 and COX-2 through metabolism by the oxidation function of these isoenzymes. This leads to inhibition of phenoxyl radical formation from a vital tyrosine residue essential for the cyclooxygenase activity of COX-1 and COX-2 and prostaglandin (PG) synthesis. Acetaminophen shows property for inhibition of the synthesis of PGs and connected factors when small amounts of Arachidonic acid and Peroxides are available but on the other hand, it has little activity at substantial levels of arachidonic acid and peroxides. The result is that Acetaminophen does not suppress the severe inflammation of rheumatoid arthritis and acute gout however will inhibit the lesser inflammation ensuing from extraction of teeth and is additionally active in a various inflammatory tests in experimental animals.
Acetaminophen typically seems to own COX-2 property, the apparent COX-2 property of action of Acetaminophen is shown by its poor anti-platelet activity and high-quality gastrointestinal tolerance. Unlike both non-selective NSAIDs and selective COX-2 inhibitors, Acetaminophen inhibits other peroxidase enzymes as well as myeloperoxidase. Inhibition of myeloperoxidase involves Acetaminophen oxidation and concomitant reduced formation of halogenating oxidants (e.g. hypochlorous acid, hypobromous acid) that will be related with multiple inflammatory pathologies as well as atherosclerosis and rheumatic diseases. It might, therefore, slow the growth of these diseases. Acetaminophen, NSAIDs and selective COX-2 inhibitors all have central and peripheral effects. As is the case with the NSAIDs, together with the selective COX-2 inhibitors, the analgesic behavior of Acetaminophen is reduced by inhibitors of several endogenous neurotransmitter systems including serotonergic, opioid and cannabinoid systems. There is considerable debate about the hepatotoxicity of therapeutic doses of Acetaminophen, most of the toxicity might result from overuse of combinations of Acetaminophen with opioids which are extensively used, mainly in USA.
- Dextromethorphan Hydrobromide
- General and Physical
Dextromethorphan HBr  is an antitussive. Dextromethorphan (DXM or DM) is a morphinan class of drug with sedative, dissociative, and stimulant properties (usually at higher doses). It is employed as cough suppressant in many ‘over the counter’ cold and cough medicines .The chemical name of Dextromethorphen Hydrobromide “ (+)-3-methoxy-17-methyl-9α,13α,14α-morphinan ”. The molecular formula as trihydrate is C18H26BrNO, molecular weight is 352.31 g/mol, it is soluble in water, the melting range is between 168ºC.
Figure 4.7 Chemical structure of Dextromethorphan Hydrobromide
- Mechanism of action
Methyl analog of ‘Dextrorphan’ that show strong affinity binding to various regions of the brain, together with the medullary cough center. This molecule is an N-Methyl-D-Aspartate (NMDA) receptor antagonist, and acts as a non-competitive channel blocker. Dextromethorphan hydrobromide is one of the broadly used Antitussives, and is also employed to study the connection of glutamate receptors in neurotoxicity.
- Dosage, Administration and pharmacology [105, 155, 157]
Dextromethorphan HBr is administered orally in different strengths as Tablets, Capsules and Oral solutions. It is administered in combination of Acetaminophen, Phenylephirine HCl, Quinidine Sulfate, Brompheniramine maleate, Guaifenesin, Pseudoephedrine, Dextromethorphan, Chlorpheniramine, Diphenhydramine, Doxylamine, Promethazine HCl. There are number of dosage strengths are available in ‘over the counter’ medicine, the usual maximum adult dose for cough in combination of Guaifenesin is 30mg in extended release tablets for every 12 hours. In oral solutions and Capsules is 20mg. The usual maximum dosage in pediatric use is 10mg and not to exceed 15mg per day.
Dextromethorphan is quickly absorbed from the gastrointestinal tract, where it enters the bloodstream and crosses the blood brain barrier, at therapeutic doses, Dextromethorphan acts centrally as opposed to locally means on the respiratory tract. It elevates the verge for coughing, without hindering ciliary activity. Dextromethorphan is quickly absorbed from the gastrointestinal tract and transformed into the active metabolite Dextromethorphan in the liver by the cytochrome P450 enzyme CYP2D6. The typical dose needed for effective antitussive treatment is between 10 and 45 mg, depending on the individual. The International Society for the Study of Cough recommends “an adequate 1st dose of medication is 60 mg in the adult and repeat dosing should be infrequent instead of the QDS recommended.
The length of action after oral administration is about 3-8 hours for Dextromethorphan-hydrobromide, and 10 – 12 hours for Dextromethorphan-polistirex. Around one in ten of the Caucasian population has very little or no CYP2D6 enzyme activity, resulting in lasting high drug levels.
- Phenylephrine Hydrochloride
- General and Physical
Phenylephrine Hydrochloride  is an alpha-1 adrenergic agonist administered as a mydriatic, nasal decongestant, and cardiotonic agent. Phenylephrine is a selective α1-adrenergic receptor agonist of the phenethylamine class administered majorly as a decongestant. It as an agent to enlarge the pupil, and to increase blood pressure. Phenylephrine is commercially marketed as an alternative for the decongestant pseudoephedrine. Although clinical trials demonstrate phenylephrine taken orally at the recommended dose, to be no more effective than placebo. Phenylephrine could also cause heart beat rate reduction in through reflex bradycardia. The chemical name is “3 – [(1R)-1-hydroxy-2-(methylamino) ethyl] phenol; hydrochloride”. The molecular formula as trihydrate is C9H14HClNO2, molecular weight is 203.67 g/mol, it is soluble in water, the melting range is between 169-172ºC.
Figure 4.8 Chemical structure of Phenylephrine Hydrochloride
- Mechanism of action
Phenylephrine hydrochloride is the acidic form of phenylephrine, which is a direct acting sympathomimetic amine. phenylephrine is chemically related to adrenaline and ephedrine with powerful vasoconstrictor behavior. Phenylephrine is a post-synaptic alpha-adrenergic receptor agonist that causes vasoconstriction and increases systolic or diastolic pressures, reflex bradycardia, and stroke output.
- Dosage Administration and pharmacology [105, 155, 158]
Phenylephrine HCl is administered orally in different dosage forms such as Tablets, Inhalers, Injections and Oral solutions. It is administered in combination of Acetaminophen, Codeine phosphate, Ketorolac, Tromethamine, Promethazine HCl, Ibuprofen, Guaifenesin, Pseudoephedrine, Chlorophenriamine maleate etc. For Phenylephrine, a variety of strengths are available in over the counter medicine. For Nasal Congestion treatment Phenylephrine given in Adults usually at 10 to 20 mg orally for every 4 hours. The usual Pediatric dose for Hypotension is 0.1 mg/kg every 1 to 2 hours as need and the maximum dose is 5mg.
Pharmacology [158, 161, 162, 163]
An in-depth investigation of the pharmacokinetics of Phenylephrine(PE) and its metabolites was reported in 1993 by Gumbhir . After oral administration of Comhist® tablets containing 10 or 20 mg of PE, reported concentrations of parent-PE in plasma were below the limit of quantitation of 2 ng/ml and the concentrations of m-hydroxymandelic acid were not detectable for the 10 mg dose. m-Hydroxymandelic acid is not extensively conjugated, whereas m-hydroxyphenylglycol is extensively conjugated. The plasma concentrations of Phenylephrine conjugates were the highest, followed by m-hydroxymandelic acid, m-hydroxyphenylglycol conjugates and m-hydroxyphenylglycol.
The pharmacokinetics of PE and its major metabolites were studied by Hengstmann and Goronzy163. Approximately 1 mg of 3 H-Phenylephrine free base was administered as an intravenous (iv) infusion over 12.5 to 20 minutes (mean 0.84 mg ± standard deviation 0.17 mg) and as an oral solution (0.99 ± 0.15 mg) to a small number of adult volunteers (N = 4 and 10, respectively). after intravenous administration, PE quickly diffuses into the peripheral tissue and which yields a very low plasma levels. Its distribution quantities during steady state ranged from 184 to 543 liters, showing most of the drug was distributed in the peripheral tissue or organs. The calculated oral PE utter bioavailability was reported as 38% relative to intravenous dosing. The biphasic distribution indicates that the drug divided into the peripheral tissue or organs upon administration, accounting for extremely low plasma concentration observed for parent PE relative to its major metabolites. The biphasic distribution of unchanged PE was confirmed by Gumbhir.
- General and Physical
Guaifenesin  is an expectorant that also contain some muscle relaxing property. It is administered in many cough preparations. The chemical name is “3-(2-methoxyphenoxy) propane-1,2-diol”. The molecular formula as trihydrate is C10H14O4, molecular weight is 198.22 g/mol, it is freely soluble in Ethanol, the melting range is between 78.5-79ºC.
Figure 4.9 Chemical structure of Guaifenesin
- Mechanism of action
Guaifenesin is an expectorant that will boost the output of phlegm (sputum) and bronchial secretions by reducing adhesion and surface tension. The improved flow of less viscous secretions promotes ciliary action and modifies a dry, unproductive cough to highly productive and less frequent. It improves the effectiveness of the mucociliary mechanism in eliminating the accumulated secretions from the higher and lower airway through decreasing the viscosity and adhesiveness of secretions.
- Dosage, Administration and pharmacology [105, 154,155, ]
Guaifenesin is administered orally in different dosage forms such as Tablets, and Oral solutions. It is administered in different strengths in combination of Acetaminophen, Hydrocodone Bitartrate, Dextromethorphan HBr, Pseudoephedrine and Chloro-pheniramine maleate etc. Usual adults immediate release strength of Guaifenesin for Cough is between 200 – 400mg for every 4 hours as needed and sustain release dose is 600 – 1200mg every 12 hours and in both cases, it should not exceed 2400mg per day. The usual pediatric dose orally is 12 mg/kg/day in six divided doses and not to exceed 600mg per day.
Pharmacology [159, 164] (Bio equivalence study)
Guaifenesin(GF) act as an expectorant, it promotes or facilitates the elimination of secretions from the respiratory tract. GF facilitates expectoration of preserved secretions through increasing mucus volume and making it less viscous.
This study characterized GF pharmacokinetics in children aged two to seventeen years (on 40 subjects). All the subjects received a single oral dose of GF (age‐based doses of 100‐400 mg) after two hours of breakfast. The blood samples were collected before and for 8 hours after dosing. Analyzed for GF using liquid chromatography‐tandem mass spectrometry, pharmacokinetic parameters were determined using noncompartmental procedures, relationships with age were assessed using linear regression, and dose proportionality was reviewed on 95% confidence intervals. Based on the upper dose recommended in the pharmacopeia (for both children and adolescents), area under the curve from time zero to infinity and Cmax both increased with age. However, similar systemic exposure was observed when comparing the higher dose for children aged 2 – 11 years with the lower dose for adolescents aged 12 – 17 years. As predicted, oral clearance (CLo) and terminal volume of distribution (Vz/F) found increasing with age this was due to increasing body size. Due to a larger increase in Vz/F than CLo, a rise in terminal exponential half‐life was additionally observed. Allometric measuring indicated no maturation‐related changes in CLo and Vz/F.
- Literature survey of simultaneous estimation analytical methods for Acetaminophen, Dextromethorphan HBr, Phenylephrine HCl and Guaifenesin
Acetaminophen: In through literature survey for Acetaminophen, the methods to estimate individually [90,125] and in combination with other compounds [122,124] are developed using liquid chromatography. A reversed-phase high-performance liquid chromatographic(HPLC) method has been developed by Sinan Suzen et.al  for the determination of Acetaminophen in pharmaceutical formulations. A ‘C18’ stationary phase column was used with a mobile phase consists of methanol & water (1/2, v/v) mixture at the column flow rate of 1.78 mL/min with the detector wavelength at 193 nm. Sulphamethoxazole is used as an internal standard and the total chromatographic run time was 5 minutes. The method has proved for linearity, precision and reproducibility.
One of the few methods reported in combination was by Ravisankar.S et.al  via reversed-phase HPLC method; it was developed for the simultaneous estimation of Acetaminophen, Ibuprofen and chlorzoxazone in formulations. The method was carried out on a Kromasil® C8 column using a mixture of 0.2% Triethylamine: Acetonitrile ( pH 3.2 using dilute orthophosphoric acid), and detection wavelength was carried out at 215 nm, “Ketoprofen” is used as internal standard. All these drugs showed linearity in the range of 2–10 μgmL−1, and limits of quantification (LOQ) was found to be 10, 50 and 20 ng ml−1 for Acetaminophen, Ibuprofen and Chlorzoxazone, respectively.
Dextromethorphan HBr: The Dextromethorphan HBr literature survey yielded few methods developed using liquid chromatography [96,97,122,123,129,130] , from those a reversed phase, combination, HPLC method was developed by Vishal Jain et.al . The method was developed for the simultaneous estimation of Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan Hydrobromide and Guaifenesin in tablet dosage form. The chromatographic conditions were established on Chromatopak C18 (25 cm × 4.6mm i.d. × 5μm) column with UV detection at 265 nm, and the mobile phase consisted of Methanol:Acetonitrile:0.025 M phosphate buffer (50:25:25, v/v/v). The retention times of Bromhexine hydrochloride, Chlorpheniramine maleate, Dextromethorphan Hydrobromide and Guaifenesin were found at 16.2 min, 12.2 min, 6.1 min and 9.4 min, respectively. The method found linear with correlation coefficients of 0.9987, 0.9988, 0.9981 and 0.9981 over a concentration range of 4.0–24.0 μg/ml for bromhexine hydrochloride, 5.0–30.0 μg/ml for chlorpheniramine maleate, and 10.0–60.0 μg/ml for both Dextromethorphan Hydrobromide and Guaifenesin, respectively. The method has validated according to the ICH guidelines and it was successfully applied to estimate the levels of four drugs in a combined formulation with good accuracy and precision.
One of the few methods reported is developed by Palled Mahes et.al97 is Reverse Phase HPLC method, it has been developed for the determination of Acetaminophen, Caffeine, Phenylephrine Hydrochloride and Dextromethorphan Hydrobromide in tablet formulation. The compounds were resolved by using a gradient mobile phase (Sodium salt of heptane sulphonic acid buffer solution and acetonitrile) at a flow rate of 1mL/min using Inertsil C8 (4.6 mm x 15 cm, 5 µm) column at a detector wavelength of 214 nm. The retention times were found to be 5, 6, 10 and 20 minutes for Acetaminophen, Caffeine, Phenylephrine HCl and Dextromethorphan HBr respectively. The percentage recovery of Acetaminophen, Caffeine, Phenylephrine Hydrochloride and Dextromethorphan Hydrobromide were observed to be in between 98% to 102%. The developed method was precise, accurate and reproducible.
Another, one of the few methods reported is developed by Zahid A. Chaudhary et.al . As per this, a Reverse Phase-HPLC method has been developed and validated for estimation of Dextromethorphan Hydrobromide and Quinidine Sulphate in combined capsule dosage form and it can be used in routine analysis. Reverse Phase-HPLC, method was carried out by isocratic technique on a reversed-phase: Thermo scientific ODS C18 (250mm X 4.6mm i.d., 5μm particle size) column and UV detection wavelength at 234 nm with mobile phase containing a mixture of Phosphate buffer (pH 4.5): Methanol (55: 45 v/v) at a flow rate of 1.0 mL/min. The retention times for Dextromethorphan Hydrobromide and Quinidine Sulphate were 3.6 and 5.5 min respectively. The method found linear in the concentration range between 10-30 μg/mL and 5-15μg/mL for Dextromethorphan Hydrobromide and Quinidine Sulphate respectively, correlation co-efficient was found to be 0.995 and 0.997 for Dextromethorphan Hydrobromide and Quinidine Sulphate respectively. In precision the average assay was found to be 99.5% and 97.00% for Dextromethorphan Hydrobromide and Quinidine Sulphate respectively. The developed method was validated as per ICH guideline, for its accuracy, precision, LOD & LOQ and the results were found to be satisfactory. The method found to be specific, rapid and simple with good sensitivity for estimation of Dextromethorphan Hydrobromide and Quinidine Sulphate. These analytical methods are also valid in ordinary laboratories, it can also be adopted for quality control tests for these drugs in capsule.
Phenylephrine HCl: In literature survey, few methods [99,100,101,126,127,128] were resulted for Phenylephrine HCl. A reversed phase HPLC method was developed by Nora.H et.al . Four different methods were developed for the concurrent estimation of Phenylephrine Hydrochloride and Chlorpheniramine Maleate without previous separation. In the first method the compounds were estimated using first derivative UV spectrophotometry, with zero-crossing measurement. The second method is based on first derivative of the ratios spectra. The third method describes the use of multivariate spectrophotometric calibration for the simultaneous determination of the analyzed binary mixture, where the resolution is obtained by using partial least squares (PLS) regression analysis. In the fourth method (HPLC), a reversed-phase column and a mobile phase consists of Methanol: Water: Acetonitrile (80:12:8 v/v/v) at 0.9 ml/min flow rate have been used to separate both drugs with a UV detection wavelength at 270 nm. All the proposed methods are thoroughly validated; and they have the advantage to be economic and time saving. The results obtained using the proposed methods are statistically analyzed and compared with some reported methods; all the described analytical methods can be used for analysis of pharmaceutical formulations.
One of the few methods reported is developed by Hamide cenyuva and Tuncel Ozden  which is Reverse Phase HPLC method described for the simultaneous estimation of Paracetamol, Phenylephrine HCl, and Chlorpheniramine maleate in combined pharmaceutical dosage forms. The method involves the mixture of acetonitrile and phosphate buffer (pH 6.22, 78:22) as the mobile phase and use of a µBondapak CN RP analytical column (125 Å, 10 µm, 3.9 × 150 mm) at 22°C as the stationary phase. Derivatization of the drugs is not required. The method can be used for commercial pediatric cough–cold syrups, tablets, and capsules marketed in Turkey. The relative standard deviation for ten replicate measurements of each drug in the medicaments is found always less than 2%.
Additionally, one of the few methods reported is developed by Khushbu B. Patel et.al . As per this, a stability indicating reversed-phase high-performance liquid chromatographic method was developed for the simultaneous estimation of ciprofloxacin hydrochloride and phenylephrine hydrochloride using a mobile phase consists of Water : Acetonitrile : Triethylamine (85: 15: 0.1, v/v/v) pH 3 adjusted with orthophosphoric acid and on a Zorbax Bonus RP C18 column. The chromatographic retention times (RT) of Ciprofloxacin Hydrochloride and Phenylephrine Hydrochloride were found to be 3.7min and 2.1min, respectively. The method linearity was established for ciprofloxacin hydrochloride and phenylephrine hydrochloride in the range of 150-900 μg/ml and 5-30 μg/ml, respectively. The % recoveries of Ciprofloxacin Hydrochloride and Phenylephrine Hydrochloride were found between 98.0-101.0%. In specificity, both the drugs were subjected to acid and base hydrolysis, oxidation, UV and thermal degradation conditions. Degradation peaks were well resolved from the main peak of drug. This method can be successfully applied for simultaneous quantitative analysis of ciprofloxacin hydrochloride and phenylephrine hydrochloride in bulk drugs and formulations.
Guaifenesin: In literature survey, few methods were yielded for Guaifenesin [102, 131,132,133]. A reversed phase HPLC method was developed by Wilcox ML et.al , for the simultaneous determination of Guaifenesin, Pseudoephedrine and Dextromethorphan in commercially available capsule dosage forms and guaifenesin-codeine in a commercial cough syrup dosage form. The separation and estimation are achieved on a 25-cm underivatized silica column using a mobile phase consists of 60:40% (v/v) 6.25 mM phosphate buffer with pH 3.0 and Acetonitrile, the column flow rate applied at 1.0 mL / min with wavelength detection at 216 nm. The chromatographic separation was achieved within 10 min for each drug mixture. The method showed satisfactory linearity for the Guaifenesin, Pseudoephedrine, Dextromethorphan mixture in the 50-200, 7.5-30 and 2.5-10 μg/ml ranges, respectively. The intra and inter-day RSDs ranged from 0.23 to 4.20%, 0.18 to 2.85%, and 0.13 to 5.04% for Guaifenesin, Pseudoephedrine, and Dextromethorphan, respectively. The Guaifenesin, Pseudoephedrine mixture yielded linear ranges of 25-100 and 3.75-15 μg/ml and intra- and inter-day RSDs ranged from 0.65 to 4.18% and 0.23 to 3.00% for Guaifenesin and Pseudoephedrine, respectively. The method showed linearity for the Guaifenesin and Codeine mixture in the 25-100 and 2.5-10 μg/ml ranges and RSDs ranged from 0.37 to 4.25% and 0.14 to 2.08% for Guaifenesin and Codeine, respectively
One of the few methods reported is developed by Abdil Ozdemir et.al . As per this a reverse phase high-performance liquid chromatographic method with fluorometric detection for the determination of Guaifenesin and Dextromethorphan in cough syrup was described. The developed method contains accurate sample preparation steps for the separation and simultaneous estimation of active compounds in samples with existence of other excipients. Amilorid (AM) was used as an internal standard (IS) since it has a fluorescence character in the working wavelength region. The chromatographic separation of two drugs and IS was achieved on a Waters Symmetry ® C18 Column 5 µm, 4.6 × 250 mm with mobile phase consisting of phosphate buffer (0.2 M, pH=2), acetonitrile and Methanol (v/v, 62:23:15). Analytes were monitored by fluorescence detection at the excitation and emission wavelengths of 277 and 588 nm, respectively. Under the established conditions, the obtained calibration graphs are found linear over the concentration range of 0.05–0.2 and 1.2–2.4 mg/mL for Dextromethorphan and Guaifenesin, respectively and the regression coefficients were found greater than 0.999. The validation study was carried out inline with the ICH guidelines to prove that the new analytical method meets the fundamental criteria including selectivity, linearity, precision, accuracy and sensitivity.
Additionally, one of the few methods reported is developed by Gugulothu Sailaja, Bollikolla Hari Babu , as per this a HPLC method it is developed and validated for simultaneous quantification of Guaifenesin, Ambroxol and Loratidine in bulk and liquid dosage form. The separation and quantification was achieved using a Kromasil C8 (250 × 4.6 mm, particles 5 μm) HPLC column. Isocratic elution program with a column flow rate of 1.2 mL/min was used, and the injection volume was 10 µL. The detector was set to a wavelength of 290 nm and the column oven temperature was maintained at 30 °C. Mobile phase consists of Orthophosphoric acid (0.1%) and acetonitrile in the ratio of 60:40 v/v. Guaifenesin, Ambroxol and Loratidine were resolved with retention time of 3.045 min, 5.489 min and 13.981 min, respectively. The method was fully validated according with ICH guidelines and the results of all the validation parameters were found to be well within the acceptable limits. The calibration plots were found linear over the concentration ranges from 50-150 µg/mL, 30-90 µg/mL and 5-15 µg/mL for Guaifenesin, Ambroxol and Loratidine, respectively. The developed method was successfully applied for the quantification of Guaifenesin, Ambroxol and Loratidine in liquid dosage form. The excipient did not interfere with drug peaks.
All above methods discussed are not suitable to analyze in the combination of Acetaminophen, Phenylephrine HCl, Dextromethorphan HBr and Guaifenesin. Based on market importance author chose to develop and validate a rapid UPLC method to estimate above compounds simultaneously.
- Amoxicillin and Clavulanate Potassium Drug product and its Impurities
- General and Physical Information:
The physical, chemical properties and structures of Amoxicillin and Clavulanate Potassium are explained in detail in section 4.1 and 4.4. The structures of impurities present in Amoxicillin and Clavulanate potassium are shown below,
- Mechanism of Action
The mechanism of Action of Amoxicillin and Clavulanate Potassium is explained in detail in section 4.1.2 and 4.4.2.
- Dosage and administration 
The dosage and administration is explained in section 4.4.3. As per this Amoxicillin and Clavulanate potassium is available in Powder for Oral suspension and Tablets. Oral administration of 5 mL of 250 mg/5 mL suspension of Amoxicillin and Clavulanate Potassium or the equivalent dose of 10 mL of 125 mg/5 mL suspension provides average peak serum concentrations of 6.9 µg/mL for Amoxicillin and 1.6 µg/mL for Clavulanic acid approximately 1 hour after dosing.
On the pharmacokinetics, dosing in the fasted or fed state has minor effect of Amoxicillin, hence it could be administered without regard to meals. Absorption of Clavulanate potassium when administered with meal is higher relative to the fasted state, in one bio study, the relative bioavailability of clavulanic acid was decreased when product was administered at 30 and 150 minutes after the initiation of a high fat breakfast.
Amoxicillin and Clavulanic acid drug product neither compound is highly protein bound. Clavulanic acid is approximately twenty five percent bound to human serum and Amoxicillin approximately eighteen percent bound. Amoxicillin distributes highly into most body tissues and fluids with the exception of the brain and spinal fluid.
After two hours of oral dosing of a single 35 mg/kg dose of Amoxicillin and Clavulanic acid suspension drug product to fasting children the average concentrations of 3 µg/mL of Amoxicillin and 0.5 µg/mL of Clavulanic acid were detected in middle ear effusions.
- Metabolism and Excretion
The half life of Amoxicillin after the oral administration of Amoxicillin and Clavulanic acid drug product is 1.3 hours and that of Clavulanic acid is 1 hour.
Approximately 50% – 70% of the Amoxicillin and approximately 25% – 40% of the Clavulanic acid are excreted unchanged in urine during the first six hours after administration of a single 250 mg or 500 mg tablet of Amoxicillin and Clavulanic acid drug product.
- Literature survey
In literature survey, a reversed phase HPLC method was developed by Durga Mallikarjuna Rao Tippa, Narendra Singh . The details of the method are described in section 4.4.4, as per this method the analytes are separated along with 6-APA impurity, nicotinamide and ticarcillin at 220nm with run time of 8 minutes but the other impurities listed in present study not addressed.
Additional method developed by Abdalla Ahmed El-Shanawani  to estimate the assay of Amoxicillin and Clavulanate potassium in dosage forms including the 6-APA and p-Hydroxyphenyl glycine impurities but not addressed most of the Amoxicillin and Clavulanate potassium impurities in present study.
One more comparison study was performed by de X.-X. Zhou et.al , this is a comparison between two methods but not addressed some of the impurities in present study.
Based on literature survey there were no stability indicating method was available for estimation of Amoxicillin and Clavulanic acid impurities simultaneously, hence author choose to develop a new method.
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