1. INTRODUCTION:

Drugs have become an essential part of life. Everyone has been taking various drugs since its birth. The quality of these drugs is an essential feature as it directly affects the life of the consumer and the quality of any drug products can be judged by analyzing it only.

Quantitative analysis of the drug is an important tool to assure its quality. Quality control is an integral part of all modern industrial process and pharmaceutical industry with no exception. For assuring the quality of the drug products there is a crucial need of specific analytical methods and because drugs have direct impact on human lives so lots of care should be taken during selection of the method.

The aim of the analytical studies is to obtain quantitative and qualitative information about the compounds of interest (analyte) in a sample. Pharmaceutical formulations are formulated with more than one drug, typically referred to as combination products. These are intended to meet desired patient need by combining their therapeutic effects of two or more drugs in one product.

These combination products can present challenges to the analytical chemists responsible for the development and validation of the analytical method for their analysis.

Testing a pharmaceutical product involves chemical, instrumental and sometimes microbiological analysis. Simultaneous estimation of drugs in combination can be carried out by using spectrophotometric and spectrofluorimetric methods and some chromatographic techniques like HPLC, HPTLC, SFC, LC/MS etc.

Planner chromatography is a multistage distribution process. It is a form of liquid chromatography in which the stationary phase is supported on a planer surface rather than a column. High performance thin layer chromatography has developed to the extent that separation and quantitation can provide results that are comparable with other analytical methods such as HPLC. HPTLC is a modern separation technique which is accepted world wide as an extremely flexible, reliable, and cost efficient method. Compared to techniques like HPLC it has features like flexibility, parallel analysis of many samples, simplified sample preparation because of single used of stationary phase and possibility of multiple evaluation of the plate with different parameters because all fractions of the sample are stored on the plates. HPTLC technique is most suited technique for content uniformity test and impurity profiling of the drugs as per compendial specification.

1.1 Parkinson’s disorder overview^[1-3]

Parkinsonism is a disturbance in motor functions characterized by rigidity, expressionless faces, stopped posture, gait disturbances, slowing of voluntary movements, and a characteristic “pill-rolling” tremor. It is not a single disease but rather is clinical manifestation of a disturbance in the dopaminerigc pathways connecting the substantia nigra to the basal ganglia. Normally, brain cells of the substantia nigra communicate to another region of the brain known as the striatum via a chemical messenger called dopamine. In Parkinson’s disease (PD), cell loss in the substantia nigra results in declining levels of available dopamine.

1.1.A. Morphology: -

Brain may be externally normal. The substantia nigra and locus ceruleus are depigmented in most cases as a result of loss of melanin containing neurons in substantia nigra, locus ceruleus and dorsal motor nucleus of vagus nerve.

1.1.B. Clinical feature:-

The cause of disease is a steady progression, over a period of about 10 years. Dementia may occur in minority of cases which is associated with presence of levy bodies in cerebral cortical neurons.

1.1.C. Signs and Symptoms:-

Parkinson’s disease is common movement disorder which is characterized by the following primary motor symptoms:

• Bradykinesia (slowed movement)

• Muscle rigidity (stiffness)

• Resting tremor (shaking; usually more pronounced on one side of the body)

• Postural instability (poor balance)

Secondary symptoms can include:

• Micrographia (small handwriting)

• Dysarthria (soft, muffled speech)

• Reduced arm swing on the affected side of the body

• Short-stepped or shuffling gait

• Reduced eye blinking and frequency of swallowing

• Depression and anxiety

• Sleep disorders

• Low blood pressure

• Constipation

1.1.D. Management:-

Levodopa combined with a peripheral acting dopa-decarboxylase inhibitor provides the mainstay of treatment in PD.

Other agents include:

· Anticholinergic drugs – Trihexyphenidyl

· Dopamine receptor agonists – Bromocriptine, Lisuride, Cabergoline, Ropinirole, Perogolide, Pramipexole

· COMT inhibitors – Entacapone

· Dopamine releasing agents – Amantadine

· MAO inhibitors – Selegiline.

Table 1. Marketed formulations of Levodopa and Carbidopa^[4]

Brand name	Company	Strength (mg)
Brand name	Company	Levodopa	Carbidopa
LCD Tablets	Intas biopharmaceuticals	50	200
		10	100
		25	100
		25	250
Levopa-C Tablets	Wallace pharmaceuticals	25	250
Neocare Tablets	VHB pharmaceuticals	10	100
		75	250
		25	100
Pardopa Tablets	Micro Synchro pharmaceuticals	10	100
		25	100
		25	250
Parkimet Tablets	GSK pharmaceuticals	25	250
Parkimet Tablets	GSK pharmaceuticals	25	100
Syndopa Tablets	Sun pharmaceuticals	10	100
		25	100
		25	250
CR-tablets		50	200
Tidomet	Torrent pharmaceuticals	25	250
		10	100
		25	100
CR-tablets		50	200

1.2. DRUG PROFILE ^[5-14]

1.2.1. LEVODOPA: -

Synonyms:

3,4-dihydroxyphenylalanine

DOPA

L-DOPA

L-Dihydroxyphenylalanine

1.2.1.A. PHYSICOCHEMICAL PROPERTIES:

Chemical formula : C₉H₁₁NO₄

IUPAC name: (2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid

Molecular weight: 197.19

Melting point: 276-278 ⁰C

CAS registry no. 59-92-7

Official status: Official in IP, BP, USP and EP.

Category: Antidyskinetic

Description: White or slightly cream, crystalline powder; odorless.

Dose: 125 to 500 mg daily, in divided doses after meals, increasing gradually in accordance with the needs of the patient; optimal dose, 1 to 8 g daily. Usually used in combination with Carbidopa.

Solubility: Slightly soluble in water; practically insoluble in chloroform, in ethanol (95%) and in ether. Freely soluble in 1M hydrochloric acid but sparingly soluble in 0.1M hydrochloric acid. The solubility of levodopa in water is 66 mg in 40 mL.

Storage: Store in well-closed, light-resistant containers.

Other properties

In the presence of moisture, levodopa is oxidized by atmospheric oxygen and darkens.

1.2.1. B. MECHANISM OF ACTION/ EFFECT:

Normal motor function depends on the synthesis and release of dopamine by neurons projecting from substantial nigra to corpus striatum. The progressive degeneration of these neurons that occurs in Parkinson's disease disrupts the nigrostriatal pathway and results in diminished levels of the intrasynaptic neurotransmitter dopamine. Striatal dopamine levels in symptomatic Parkinson's disease are decreased by 60 to 80%, Striatal dopaminergic neurotransmission may be enhanced by exogenous supplementation of dopamine through administration of dopamine's precursor, levodopa. A small percentage of each levodopa dose crosses the blood-brain barrier and is decarboxylated to dopamine. This newly formed dopamine then is available to stimulate dopaminergic receptors, thus compensating for the depleted supply of endogenous dopamine.

1.2.1.C. PHARMACOKINETICS:

Absorption:

Levodopa is rapidly absorbed from the proximal small intestine by the large neutral amino acid (LNAA) transport carrier system.

This transport system is a saturable, sodium-independent, facilitated mechanism for aromatic and branched chain amino acids. The capacity of the transport system is limited and levodopa must compete for energy-dependent proximal small bowel absorption sites.

Stomach and intestinal walls contain abundant levels of the L-aromatic amino acid decarboxylase (AAAD) enzyme, which degrades levodopa and thus serves as a significant barrier to the absorption of intact levodopa; only about 30% of an orally administered dose reaches the circulation as intact levodopa.

Absorption may be enhanced by concomitant administration of a peripheral decarboxylase inhibitor, such as carbidopa or a catechol- O-methyltransferase (COMT) inhibitor, such as tolcapone. With long-term administration, levodopa absorption appears to become more efficient and complete.

High gastric acidity, delayed stomach emptying time, and the presence of certain other amino acids, such as those that occur after digestion of a protein-containing meal, may prevent absorption of levodopa. Intense exercise and other activity that diverts blood flow from the mesenteric circulation also may delay levodopa absorption.

Distribution:

Levodopa is widely distributed to most body tissues, but not to the central nervous system (CNS) because of extensive metabolism in the periphery. Levodopa crosses biological membranes, including the intestinal epithelium and the blood-brain barrier, by means of the LNAA transport system. This system is the saturable, stereospecific, facilitated transport mechanism for large neutral amino acids, including those from dietary protein intake. The transport rate across the blood-brain barrier is dependent upon the plasma concentration of levodopa and the concentration of competing amino acids. The flux of amino acids across the blood-brain barrier is bidirectional; the net flux of unmetabolized levodopa is from the brain into the plasma as levodopa plasma concentrations fall.

Metabolism:

95% of an administered oral dose of levodopa is pre-systemically decarboxylated to dopamine by the L-aromatic amino acid decarboxylase (AAAD) enzyme in the stomach, lumen of the intestine, kidney, and liver. This converted portion of dopamine cannot cross the blood-brain barrier to exert its effects on the brain. Dopamine remaining in the periphery is believed responsible for many levodopa adverse effects, including cardiac arrhythmias and gastrointestinal upset. Levodopa also may be methoxylated by the hepatic catechol- O-methyltransferase (COMT) enzyme system to 3- O-methyldopa (3-OMD), which cannot be converted to central dopamine. 3-OMD has a long half-life and competes with levodopa for the same transport mechanism across the blood-brain barrier.

When the portion of the remaining intact levodopa does cross the blood-brain barrier, it is decarboxylated to dopamine, which is normally stored in presynaptic terminals of dopaminergic neurons in the striatum. After release into the synapse, dopamine is transported back into the dopaminergic terminals by the presynaptic uptake mechanism, or is further metabolized by monoamine oxidase (MAO) or COMT. The effects of levodopa in the brain are affected by the rate and extent of cerebral conversion to dopamine, the rate of movement of the synthesized dopamine to the striatal receptors, and the rate of inactivation of newly synthesized dopamine.

Half-life:

Levodopa: 0.75 to 1.5 hours.

3-O-methyldopa (3-OMD): 15 hours; accumulation will occur during chronic dosing.

Onset of action:

Significant improvement may occur in 2 to 3 weeks. Some patients may require up to 6 months of continuous levodopa therapy to obtain optimal therapeutic benefit.

Time to peak concentration:

0.5 to 2 hours.

Elimination:

Renal, 70 to 80% of dose eliminated within 24 hours, largely as dopamine metabolites. Homovanillic acid (HVA) is a major urinary metabolite, accounting for 13 to 42% of the ingested dose of levodopa in twenty-four hour urine samples. Unchanged levodopa accounts for less than 1% of an administered dose. Some of the eliminated metabolites may color the urine red; oxidation that occurs when urine is exposed to air will cause it to darken. Fecal, 2% of dose.

1.2.1.D. GENERAL CONSIDERATIONS:

Although levodopa is the most effective antiparkinsonian medication and remains a mainstay of therapy for symptomatic treatment of Parkinson's disease, complications to long-term levodopa therapy appear commonly. The majority of patients receiving chronic levodopa therapy experience serious adverse effects, including motor fluctuations, dyskinesias, and neuropsychiatric effects Fluctuations in response to levodopa therapy represent a significant problem in the long-term management of patients with Parkinson's disease. Later stage motor complications are related to the severity and duration of the underlying disease, as well as to treatment-related factors such as the duration and dose of levodopa therapy Patients who develop response fluctuations to levodopa therapy appear to lack the capacity to buffer fluctuations in plasma levels of levodopa Therapeutic response to levodopa therapy includes a short-duration response, in which improvement in motor disability lasts for a few hours after the administration of a single dose of levodopa, and a long-duration response, in which antiparkinsonian effects may last for many hours or days following discontinuation of levodopa.

1.2.1.E. SIDE/ADVERSE EFFECTS:

Complications to long-term levodopa therapy appear commonly and include motor fluctuations, dyskinesias, and neuropsychiatric problems

Neuropsychiatric effects may occur in up to two-thirds of patients on long-term levodopa therapy and may be related to the activation of dopamine receptors in nonstriatal regions of the brain, especially the cortical and limbic regions. These mental and behavioral changes include confusion, agitation, hallucinations, irritability, panic, paranoid delusions, mental depression, dementia, mania, and psychosis; euphoria, hypersexuality.

1.2.2. CARBIDOPA: -

Synonyms:

Alpha-Methyldopahydrazine

Carbidopa Anhydrous

Carbidopa Monohydrate

Carbidopum [INN-Latin]

N-Aminomethyldopa

1.2.2.A. PHYSICOCHEMICAL PROPERTIES:

Chemical formula : C₁₀H₁₄N₂O₄,H₂O

IUPAC name : (S)-2-(3,4-dihydroxybenzyl)-2-hydrazino- propionic acid monohydrate.

Mol. Wt. 244.25

Melting Point: 203-205 ⁰C

CAS registry no. 28860-95-9

Official status: Official in IP, BP, USP and EP.

Category: Antiparkinsonian with Levodopa.

Description: White to creamy white powder; odorless or practically odorless.

Dose: 10 to 25 mg in combination with Levodopa.

Solubility: Slightly soluble in water; very slightly soluble in ethanol (95%) and in methanol; practically insoluble in acetone, in chloroform, in dichloromethane and in ether. It is soluble in dilute solutions of mineral acids.

Storage: Store in well-closed, light-resistant containers.

Indication : For treatment of the symptoms of idiopathic Parkinson's disease (paralysis agitans), post-encephalitic Parkinsonism.

1.2.2.B. MECHANISM OF ACTION:

Carbidopa inhibits decarboxylation of peripheral levodopa. It does not cross the blood-brain barrier readily and does not affect the metabolism of levodopa within the central nervous system at doses of carbidopa that are recommended for maximum effective inhibition of peripheral decarboxylation of levodopa.

Since its decarboxylase-inhibiting activity is limited primarily to extracerebral tissues, administration of carbidopa with levodopa makes more levodopa available for transport to the brain.

1.2.2.C. PHARMACOKINETICS:

Carbidopa reduces the amount of levodopa required to produce a given response by about 75% and, when administered with levodopa, increases both plasma levels and the plasma half-life of levodopa, and decreases plasma and urinary dopamine and homovanillic acid.

The introduction of carbidopa to levodopa therapy, which inhibits the peripheral decarboxylation of levodopa to dopamine, counteracts the metabolic-enhancing effect of pyridoxine.

Absorption: Rapidly decarboxylated to dopamine in extracerebral tissues so that only a small portion of a given dose is transported unchanged to the central nervous system.

Protein binding: 76%

Biotransformation: Rapidly decarboxylated to dopamine.

Half life: 1-2 hours..

1.2.3. ADVERSE REACTIONS OF COMBINATION THERAPY:

CNS: anxiety, dizziness, hallucinations, memory loss, headache, numbness, confusion, insomnia, nightmares, delusions, psychotic changes, depression, dementia, poor coordination, worsening hand tremor

CV: cardiac irregularities, palpitations, orthostatic hypotension

ENT: blurred vision, diplopia, mydriasis, eyelid twitching, difficulty swallowing

GI: nausea, vomiting, diarrhea, constipation, abdominal pain or discomfort, flatulence, excessive salivation, dry mouth, anorexia, upper GI hemorrhage (with history of peptic ulcer)

GU: urinary retention, urinary incontinence, dark urine

Hematologic: hemolytic anemia, leucopenia

Hepatic: hepatotoxicity

Musculoskeletal: muscle twitching, involuntary or spasmodic movements

Respiratory: hyperventilation

Skin: melanoma, flushing, rash, abnormally dark sweat

Other: altered or bitter taste, burning sensation of tongue, tooth grinding (especially at night), weight changes, hot flashes, hiccups.

1.2.4. INTERACTIONS:

Drug-drug. Anticholinergics: decreased carbidopa-levodopa absorption

Antihypertensives: additive hypotension

Haloperidol, papaverine, phenothiazines, phenytoin, reserpine: reversal of carbidopa-levodopa effects

Inhalation hydrocarbon anesthetics: increased risk of arrhythmias

MAO inhibitors: hypertensive reactions

Methyldopa: altered efficacy of carbidopa-levodopa, increased risk of adverse CNS reactions

Pyridoxine: antagonism of carbidopa-levodopa effects

Selegiline: increased risk of adverse reactions

1.2.5. CONTRAINDICATIONS:

• Hypersensitivity to drug or tartrazine, MAO inhibitor use within past 14 days

• Angle-closure glaucoma, Malignant melanoma

1.3. INTRODUCTION TO HPTLC^[15,16]

1.3.1. Principles of thin-layer chromatography:

Thin-layer chromatography (TLC), also known as planar chromatography (PC), is one of the oldest methods in analytical chemistry still in use.

In TLC, the different components of the sample are separated by their interaction with the stationary phase (bonded to the glass, aluminum, or plastic support) and the liquid mobile phase that moves along the stationary phase.

TLC is a fast, simple, and low-cost method suitable for any laboratory. A particular advantage is that it allows the analysis of many samples simultaneously. In contrast to liquid chromatography (LC), TLC offers separation without or at least with minimal sample preparation. Also, the plates are disposable, and there is no memory effect, such as may occur in LC. TLC is also an off-line method: sample application, separation, and detection take place in different processes. Because of its off-line character, TLC allows the use of a number of detection methods and appropriate derivatization reagents in sequence, which improves the reliability of the detection.

Table 2. Difference between HPTLC and TLC:-

Parameters	HPTLC	TLC
Layer of Sorbent	100µm	250µm
Efficiency	High due to smaller particle size generated	Less
Separations	3 - 5 cm	10 - 15 cm
Analysis Time	Shorter migration distance and the analysis time is greatly reduced	Slower
Solid support	Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes	Silica gel , Alumina and Kiesulguhr
Development chamber	New type that require less amount of mobile phase	More amount
Sample spotting	Auto sampler	Manual spotting
Scanning	Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer	Not possible

1.3.2. Features of HPTLC:

Ø Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard

Ø Several analysts work simultaneously

Ø Lower analysis time and less cost per analysis

Ø Low maintenance cost

Ø Simple sample preparation - handle samples of divergent nature

Ø No prior treatment for solvents like filtration and degassing

Ø Low mobile phase consumption per sample

Ø No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination

Ø Visual detection possible - open system

Ø Non UV absorbing compounds detected by post-chromatographic derivatization

1.3.3. Steps involved in HPTLC:

1. Selection of chromatographic layer

2. Sample and standard preparation

3. Layer pre-washing

4. Layer pre-conditioning

5. Application of sample and standard

6. Chromatographic development

7. Detection of spots

8. Scanning

9. Documentation of chromatic plate

Selection of chromatographic layer

Precoated plates with different support materials and different Sorbents are available 80% of analysis is done on silica gel GF.

Activation of pre-coated plates

Plates exposed to high humidity or kept on hand for long time requires activation.

Activation of pre-coated plates is done by placing them in an oven at 110-120ºc for 30’ prior to spotting. Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes.

Application of sample and standard

Usual concentration range is 0.1-1µg / µl

Above this causes poor separation automatic applicators are available wherein nitrogen gas sprays sample and standard from syringe on TLC plates as bands

Band wise application provides better separation and shows high response to densitometer

Selection of mobile phase

- Trial and error

- One’s own experience and Literature

- When the mobile phase is polar, polar compounds would be eluted first because of lower affinity with stationary phase

- Non-Polar compounds retained because of higher affinity with the stationary phase for chromatographic development twin trough chambers are used where only 10 -15 ml of mobile phase is required

- Components of mobile phase should be mixed thoroughly and then introduced into the twin trough chamber

Pre- conditioning (Chamber saturation)

Unsaturated chamber may lead to high Rf values

Saturation of chamber is done by lining with filter paper for 30 minutes prior to development.

This allows uniform distribution of solvent vapors in the chamber so less solvent is required for the sample to travel.

Chromatographic development and drying

After development, plate is removed from the chamber and mobile phase is removed from the plate. Drying can be done either at room temperature or at elevated temperatures if solvents like water or acids are used.

Detection and visualization

Detection under UV light gives benefit of non destructiveness.

Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length)

Spots of non fluorescent compounds can be seen when fluorescent stationary phase is used like as silica gel GF.

Non UV absorbing compounds can be visualized by dipping the plates in 0.1% iodine solution. When individual component does not respond to UV–derivatisation is required for detection.

Quantification
Sample and standard are chromatographed on same plate. After development of the plate chromatogram is scanned.

TLC scanner scans the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode. Scanning speed is selectable up to 100 mm/s and spectra recording are fast - 36 tracks with up to 100 peak windows can be evaluated.

Calibration of single and multiple levels with linear or non-linear regressions are possible. When target values are to be verified such as stability testing and dissolution profile single level calibration is suitable.

Statistics such as RSD or CI are reported automatically.

Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors.

1.4. INTRODUCTION TO VALIDATION OF METHOD^[17-18]

1.4.1. Linearity

The linearity of an analytical method is its ability to elicit test results that are directly or by a well-defined mathematical transformation proportional to the concentration of analyte in samples within a given range.

The range of analytical method is the interval between upper and lower level of analyte including levels that have been demonstrated to be determining with precision and accuracy using the method. Results were expressed in terms of Correlation co-efficient.

1.4.2. Precision

The precision is measure of either the degree of reproducibility or repeatability of analytical method.

It provides an indication of random error. The precision of an analytical method is usually expressed as the standard deviation, Relative standard deviation or coefficient of variance of a series of measurements.

a. Repeatability (Precision on replication):

It is a precision under a same condition (Same analyst, same apparatus, short interval of time and identical reagents) using same sample.

Repeatability of measurements at 283 nm:

8 µl aliquot of stock solution (50 µg/ml) was spotted on the TLC plate under nitrogen stream and was developed and scanned seven times without changing plate position at 283 nm in absorbance mode. % C.V. was calculated.

b. Intraday and Interday Precision:

Variation of results within same day is called Intraday precision and variation of results amongst days is called Interday precision.

Intraday precision was determined by analyzing Levodopa and carbidopa (300, 400 and 500 ng/spot) for three times in the same day and % C.V. was calculated.

Interday precision was determined by analyzing Levodopa and carbidopa (300, 400 and 500 ng/spot) daily for three days and % C.V. was calculated.

1.4.3 Accuracy

Accuracy of an analysis is determined by systemic error involved. It is defined as closeness of agreement between the actual (true) value and analytical value and obtained by applying test method for a number of times.

Accuracy may often be expressed as % Recovery by the assay of known, added amount of analyte. It is measure of the exactness of the analytical method.

It was determined by calculating the recovery of Levodopa and carbidopa by Standard addition method. To a fixed amount of levodopa and carbidopa(300 ng/spot), increasing amount of levodopa and carbidopa was added at three levels of calibration curve and the amount of both drugs were calculated at each level.

1.4.4 Limit of Detection

It is the lowest amount of analyte in sample that can be detected but not necessarily quantitated under the stated experimental conditions. It can be determined by three methods:

(I) Based on visual evaluation: It is determined by the analysis of samples with known concentrations of analyte and establishing the minimum level at which the analyte can be reliably detected.

(II) Based on signal to noise ratio: Determination of the signal to noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal to noise ratio of 2:1 or 3:1 is generally considered acceptable for estimating the detection limit.

(III) Based on standard deviation of the response and the slope: The detection limits may be expressed as:

DL = 3.3 s / S

Where, s = the standard deviation of the response

S = the slope of calibration curve

From the linearity curves of both the drugs the standard deviation of the intercept was calculated and the value obtained was substituted in the above equation to get limit of detection for both the drugs respectively.

1.4.5 Limit of Quantification

It is the lowest concentration of analyte in the sample that can be determined with the acceptable precision and accuracy condition. It can be determined by three methods:

(I) Based on visual evaluation: It is determined by the analysis of samples with known concentrations of analyte and establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision

(II) Based on signal to noise: Determination of the signal to noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably quantified. A signal to noise ratio of 10:1 is generally considered acceptable for estimating the quantitation limit.

(III) Based on standard deviation of the response and the slope: The quantitation limits may be expressed as:

DL = 10 s / S

Where, s = the standard deviation of the response

S = the slope of calibration curve

From the linearity curves of both the drugs the standard deviation of the intercept was calculated and the value obtained was substituted in the above equation to get limit of quantification for both the drugs respectively.

2. EXPERIMENTAL WORK:

2.1. IDENTIFICATION OF DRUGS:^{[38, 39]}

Identification of drugs was carried out by IR spectroscopy, melting point and U.V spectra studies.

2.1.1. Infrared Spectroscopy:

A mixture of drug samples and KBr (spectroscopic grade) was prepared using mortar-pestle. The mixture was analyzed by attenuated reflectance FT-IR. The mixture was scanned from 4000-400 cm^-1 and a spectra was recorded with the help of IR spectrophotometer (JASCO model: FT-IR 6100 Type A),(figure1, 2; table 4, 5)

Figure 1. Recorded IR spectra of levodopa

Table 7. Comparison of recorded and reported IR spectra for Levodopa

IR peak of levodopa	Reported wave number(cm^‑1)	Recorded wave number(cm^-1)
OH-stretching (bonded)	3375, 3210	3350, 3230
NH₃⁺	3070, 2700-2300	3073.98, 2364.3
COO^-	1656, 1569	1680, 1567.36
Aromatic CH out of plane bending of two adjacent free H’s	821, 816	819.47, 817.67

Figure 2. Recorded IR spectra of carbidopa

Table 8. Comparison of recorded and reported IR spectra for Carbidopa

IR peak of carbidopa	Reported wave number (cm^‑1)	Recorded wave number (cm^-1)
COO^-	1625	1629.56
N-N	1525	1527.36
NH₃⁺	2300	2345.02
NH₃⁺	875	879.38
OH^-	3500, 3620	3561.88
Tertiary carbon	920,880	916.22

The UV spectra of levodopa and carbidopa are similar to each other. This is due to the fact that the structures of both the drugs are similar to each other and contains identical chromophoric groups.

Table 9. Wavelength maxima of levodopa and carbidopa

Drug	Reported maxima (nm)	Recorded maxima (nm)
Levodopa	280	282
carbidopa	284	285

2.1.3. Melting point determination:

Melting point of levodopa and carbidopa was determined using the melting point apparatus.

The melting point of compounds were taken by open capillary method and are reported in

Table 6. Melting point data of levodopa and carbidopa

Drug	Reported melting point (⁰C)	Observed melting point (⁰C)
Levodopa	276-280	276-278
Carbidopa	204-209	206-208

2.2. Apparatus and Instruments:

Ø Pre-coated silica gel aluminum plate 60 F₂₅₄ TLC plate (5×20 cm, layer thickness 2 mm (E. Merck)

Ø Camag – Twin trough chamber (10 ´ 10) with stainless steel Lid

Ø Camag Applicator-Linomat V

Ø Camag TLC scanner 3

Ø Camag – 100 µl Applicator syringe (Hamilton, Bonaduz, Schweiz)

Ø UV cabinet with dual wavelength UV lamp

Ø Balance Model: Metler Toledo

Ø Ultra Sonicator, Syclon sonicator, Ningbo Sklon Lab

Instrument Co., Ltd India

Ø Amber coloured volumetric flask - 100ml, 50ml, and 10ml.

2.3. Reagents and Materials:

Ø Levodopa API (gifted by Alembic Research Centre, Vadodara.)

Ø Carbidopa API (gifted by Alembic Research Centre, Vadodara.)

Ø Ethanol, Baroda chemicals pvt. Ltd., Baroda

Ø 0.05 N HCl Laboratory grade, s.d. fine chemicals, Mumbai

Ø Acetone, AR grade, s.d. fine chemicals, Mumbai

Ø Chloroform, AR grade, s.d. fine chemicals, Mumbai

Ø n-butanol, AR grade, Rankem laboratories, India

Ø Glacial acetic acid (GAA), AR grade, s.d. fine chemicals, Mumbai

Ø Double distilled water

Ø Tablets containing Levodopa and Carbidopa.

2.4. Chromatographic condition:

Ø Stationary phase: Pre-coated silica gel aluminum Plate 60F–254 (E. Merck) pre-washed with Methanol then dried for 30 minute at 60°C.

Ø Mobile phase: Acetone : Chloroform: n-butanol : GAA : water (5 : 5 : 4.0 : 3.5 : 2.0, v/v/v/v/v)

Ø Chamber saturation: 30 min

Ø Band width: 3.8 mm

Ø Distance run: 60 mm

Ø Run time: 18 ± 2 minutes

Ø Scanning Wave length: 283 nm

Ø Slit width: 4 mm

Ø Slit height: 0.45 mm

Ø Evaluation mode: Absorbance

Ø Lamp: Deuterium/Tungsten

2.5. Preparation of stock solution of levodopa:

Standard levodopa (5 mg) was accurately weighed and transferred to 50 ml volumetric flask. It was dissolved properly and diluted up to mark with Ethanol: 0.05N HCl(1:1, v/v) to obtain final concentration of 100 µg/ml. Suitable aliquot of this solution was transferred in a 50 ml volumetric flask and diluted up to mark with Ethanol: 0.05N HCl(1:1, v/v) to obtain final concentration of 50 µg/ml. This solution was used as working standard solution.

2.6. Preparation of stock solution of Carbidopa:

Standard carbidopa (5 mg) was accurately weighed and transferred to 50 ml volumetric flask. It was dissolved properly and diluted up to mark with Ethanol: 0.05N HCl(1:1, v/v) to obtain final concentration of 100 µg/ml. Suitable aliquot of this solution was transferred in a 50 ml volumetric flask and diluted up to mark with Ethanol: 0.05N HCl(1:1, v/v) to obtain final concentration of 50 µg/ml. This solution was used as working standard solution.

2.7. Pre-treatment of pre-coated plates:

TLC plate was placed in twin trough glass chamber containing methanol as mobile phase. Methanol was allowed to run up to upper edge of plate (ascending method). Plate was removed and allowed to dry in oven at 50⁰C for 20 min. For the actual experiment the plate was allowed to come to room temperature and used immediately.

2.8. Calibration curve for standard levodopa and carbidopa:

From the stock solution (50 µg/ml) aliquots of 4,6,8,10,12 and 14µl were spotted on the TLC plate under nitrogen stream using Linomat V to obtain final concentration range of 200-700 ng/spot.

Standard	Application volume (µl)	Conc. Per spot (ng)
Standard 1	4	200
Standard 2	6	300
Standard 3	8	400
Standard 4	10	500
Standard 5	12	600
Standard 6	14	700

2.9. Analysis of prepared standards:

The plates were developed in Twin trough developing chamber (10 ´ 10 cm) with stainless steel Lid, previously saturated with the mobile phase for 30 min. The plates were removed from the chamber after development and were dried in Hot air oven at 60⁰C for 2 hrs.

The plates were scanned and quantified at 283 nm in absorbance mode with camag TLC scanner 3. The calibration curve was constructed by plotting peak area vs. concentration (ng/spot) corresponding to each spot and regression equation was calculated.

2.10. Quantification of Levodopa and Carbidopa in Tablet:

2.10.1 Preparation of test stock solution:

To determine the content of levodopa and carbidopa in tablet, the contents of 20 tablets were weighed and their mean weight determined and finely powdered. An equivalent weight of the tablet content (100 mg levodopa and 25 mg carbidopa) was transferred into a 100 ml volumetric flask containing 50 ml ethanol: 0.05N HCl (1: 1, v/v), sonicated for 15 min and further diluted to 100 ml with Ethanol: 0.05N HCl(1:1, v/v). The resulting solution was sonicated for 30 min and supernatant was filtered through whatman filter paper.

2.10.2 Preparation of Levodopa test solution:

2 ml of the test stock solution was taken in a 10 ml volumetric flask and was diluted up to mark with Ethanol: 0.05N HCl (1:1, v/v) to obtain final concentration of 50 µg/ml of levodopa.

2.10.3 Preparation of carbidopa test solution: 0.5 ml of the test stock solution was taken in a 10 ml volumetric flask and was diluted up to mark with Ethanol: 0.05N HCl (1:1, v/v) to obtain final concentration of 50 µg/ml of carbidopa.

2.10.4. Analysis of prepared samples:

8 µl from these solutions were spotted on the TLC plate under nitrogen stream using Linomat V. The plate was dried in air, and then the plate was developed in Twin trough developing chamber, previously saturated with the mobile phase for30 min. The plates were removed from the chamber after development and were dried in Hot air oven at 60⁰C for 2 hrs.

The plates were scanned and quantified at 283 nm in absorbance mode with camag TLC scanner 3.The concentration of sample solution was found from calibration curve of levodopa and carbidopa respectively. Recovery studies were performed by standard addition method.

2.11. RESULTS AND DISCUSSION:

2.11.1 Selection and optimization of solvent and mobile phase:

· Selection and optimization of a proper mobile phase is a challenging task in HPTLC method development. Several factors affects the selection of mobile phase such as polarity of the drugs, desired Rf values, practical problems such as diffusion of spots, tailing, proper peak shape after scanning.

· HPTLC method for identification of Levodopa and Carbidopa in mixture is official in USP with mobile phase Acetone: Chloroform: n-butanol: GAA: water (60: 40: 40: 40: 35, v/v/v/v/v), run length 15 cm. spraying reagent ninhydrin prepared in n-butanol and GAA mixture(plate heated at 105⁰C for about 10 minutes). The solvent used to prepare solutions of both the drugs is methanol: 0.05N HCl (1: 1, v/v).

Figure 5. Linearity curve for levodopa

Figure 6. Linearity curve for levodopa from winCATS software

Table 11. Calibration data of Levodopa by HPTLC with UV detection

Sr. No.	Concentration (µg/spot)	Peak Area		Rf
Sr. No.	Concentration (µg/spot)	Mean ± SD	%RSD	Rf
1	200	1730.4± 154.64	6.1	0.26
2	300	2459.6 ± 97.75	2.9	0.24
3	400	3385.4 ±136.08	3.4	0.25
4	500	4163.6 ±138.30	3.1	0.25
5	600	5036.8 ± 97.72	1.9	0.26
6	700	5670.8 ± 51.99	0.9	0.26

Table 12. Calibration data of Carbidopa by HPTLC with UV detection

Sr. No.	Concentration (µg/spot)	Peak Area		Rf
Sr. No.	Concentration (µg/spot)	Mean ± SD	%RSD	Rf
1	200	1830.4± 204.2	11.3	0.81
2	300	2450.9± 84.37	3.5	0.83
3	400	2920.8±159.53	5.2	0.83
4	500	3500.3± 63.95	1.8	0.82
5	600	3930.9±199.86	4.9	0.81
6	700	4410.1± 158.09	3.5	0.83

Figure 7. Linearity curve for carbidopa

Figure 8. Linearity curve for carbidopa from win CATS software

Table 13. Repeatability data of Levodopa by HPTLC with UV detection

Time	Peak Area	Rf
1^st	4479.2	0.23
2^nd	4512.2	0.23
3^rd	4482	0.21
4^th	4600.1	0.22
5^th	4591.2	0.24
6^th	4700.2	0.24
7^th	4554.6	0.21
Mean	4590.67	0.2285
S.D.	48.37	0.0069
%RSD	0.99	3.1

Figure 9. HPTLC chromatogram of levodopa (Rf = 0.26) carbidopa (Rf = 0.83) standard mixture.

Figure 10. HPTLC chromatogram (3D view) for linearity of levodopa and carbidopa

5.11.2.2. Precision

2.11.2.2.1. Repeatability

Table 14. Repeatability data of Carbidopa by HPTLC with UV detection

Time	Peak Area	Rf
1^st	2764.20	0.83
2^nd	2826.10	0.84
3^rd	2908.10	0.81
4^th	2792.20	0.79
5^th	2861.80	0.82
6^th	2964.20	0.84
7^th	2842.80	0.80
Mean	2851.35	0.8014
S.D.	68.15	0.0069
%RSD	2.3	0.86

2.11.2.6 Summary of Validation parameters:

Table 21. Summary of Validation parameters by HPTLC with UV detection

Sr. No	Parameters	Levodopa	Carbidopa
1	Linearity range (ng/spot)	200-700	200-700
2	Regression equation	y = 6.071x + 1346	y = 5.119x + 870.1
3	Correlation coefficient (r²)	0.997	0.997
4	Intercept	1510.5	822.97
5	Slope	5.8459	5.3597
6	Precision Intra day % RSD (n = 3) Inter day % RSD (n = 3) Repeatability of measurements % RSD	2.7 1.8 0.99	2.6 2.9 2.3
7	Limit of detection	25.5 ng	51.56 ng
8	Limit of quantification	57.56 ng	86.87

2.11.2.7. Estimation of Levodopa and Carbidopa in marketed Tablet:

The developed method was used to estimate Levodopa and Carbidopa in the tablet dosage form. Three different brands of tablet formulations were procured from the market for analysis by the proposed method. The percentage of Levodopa and Carbidopa was found from the calibration curve of the standard drug respectively.

Figure 11. HPTLC chromatogram of assay of tablet samples (500 ng/spot)

Track 1 : Standard drug mixture(500 ng/spot)

Track 2: Syndopa – Levodopa

Track 3: Syndopa – Carbidopa

Track 4: Tidomet – Levodopa

Track 5: Tidomet – Carbidopa

Track 6: LCD – Levodopa

Track 7: LCD – Carbidopa

Table 22. Estimation of Levodopa in tablet by HPTLC with UV detection

Dosage form	Brand names	Labeled Claim (mg/tablet)	Peak Area
Dosage form	Brand names	Labeled Claim (mg/tablet)	Amount found (mg/tablet)	% Recovery ± S.D
Tablet	Syndopa (Sun)	100	98.9	95.6±0.85
Tablet	Tidomet (Torrent)	100	104.1	102.55 ± 1.91
Tablet	LCD (Intas)	100	103.6	103.1± 1.84

Table 23. Estimation of Carbidopa in tablet by HPTLC with UV detection

Dosage form	Brand names	Labeled Claim (mg/tablet)	Peak Area
Dosage form	Brand names	Labeled Claim (mg/tablet)	Amount found (mg/tablet)	% Recovery ± S.D
Tablet	Syndopa (Sun)	25	26.63	104.95 ± 1.34
Tablet	Tidomet (Torrent)	25	23.15	96.1 ±1.13
Tablet	LCD (Intas)	25	23.82	96.8 ± 0.42

Table 24. Degradation of carbidopa in levodopa-carbidopa mixture.

Days	Peak Area (for 500 ng/spot)
Days	Levodopa	Carbidopa	Degradant
1^st	4334	3890	-
2^nd	4147	2814.2	190
3^rd	4049	2450	1186
4^th	3933.6	-	1915

3.0 CONCLUSION:

By developing HPTLC method, it can be concluded that High performance thin layer chromatography method is a most suitable technique for the analysis of combination of commercial formulations of levodopa and carbidopa than HPLC, LC-MS method, as it is simple and accuracy and precision is satisfactory.

The developed method is highly sensitive and specific with higher accuracy and precision makes it easy handling for routine analysis of levodopa and carbidopa from its combined dosage form. A good % recovery for both the drugs shows that the developed method is free of the interference of excipients used in the formulation. A satisfactory limit of quantification found found so that minute quantity and low doses also been detected and measured by using this method.

4.0 SUMMARY:

HPTLC method was developed for the simultaneous Estimation of Levodopa and Carbidopa in their combine dosage form. The developed was simple and reliable, easily handled method. It is validated for Accuracy, linearity, precision, repeatability, limit of detection and limit of quantitation with inter and and intraday accuracy and precision. This makes it suitable for the industry level.

The drugs showed linearity in the range of 200-700 ng/spot with correlation coefficient of 0.997 for levodopa and 0.997 for carbidopa. The method was found accurate, precise, specific, selective and repeatable. The minimum detectable concentration of Levodopa and Carbidopa was found to be 25.5 ng /spot and 51.56 ng/spot respectively. The lowest quantifiable concentration of Levodopa and carbidopa was found to be 57.56 ng/spot and 86.87ng/spot respectively.

The developed method consisted of Acetone: Chloroform: n-butanol: GAA: water (5 : 5 : 4.0 : 3.5 : 2.0, v/v/v/v/v) as mobile phase. Saturation time was kept 30 minutes with run length of 60 mm. the drugs were separated at the Rf value of 0.26 for levodopa and 0.83 for carbidopa.

The developed method was utilized for determination of the assay of tablets containing levodopa and carbidopa, of three different brands. It is accurate and shows highly accurate result.

By this method we can also analyse and separate a degradation product of the levodopa, carbidopa and mixture of both in solution mixture. So If degradation done during storage or in stability study, one can easily determined the degraded products by using this method. In industry it is useful degraded stability study.

5.0 FUTURE SCOPE

The developed HPTLC method was too easy and highly accurate as well onetime cost for establishment, so it is very useful for determination of thesedrugs in industry in very low concentrations of these drugs either alone or in combination. This method also employed for separating the degradation product formed in the solution of the drug mixture. From literature review and finding from the method it was evident that the degradant formed was due to carbidopa. Hence this method can be used to develop a stability indicating assay for carbidopa.So it it very useful in stability study of both of this drug in Industry.

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Received on 25.04.2014 Accepted on 28.05.2014

Asian J. Pharm. Ana. 4(2): April-June 2014; Page 57-77

Sr. No.	Trials	Observation	Remarks
1	n-butanol: glacial acetic acid: cyclohexane (8:1:1, v/v/v) Run length = 50mm	Improper resolution, diffused spot	Not satisfactory
2	n-butanol: glacial acetic acid: water (8: 1: 1, v/v/v) (8: 1: 2, v/v/v) (8: 2: 2, v/v/v) Run length = 70mm	Improper resolution, diffused spot	Not satisfactory
3	Toluene: ethyl acetate: glacial acetic acid (4: 2: 3.5, v/v/v/) (4: 2: 4.5, v/v/v) Run length = 65mm	Very high Rf values, diffused spots	Not satisfactory
4	Acetone: chloroform: n-butanol: glacial acetic acid: (6: 4: 4: 4, v/v/v/v) (6: 4: 4: 3.5, v/v/v/v) Run length = 65mm	Very high Rf values, diffused spots	Not satisfactory
5	Acetone: chloroform: n-butanol: glacial acetic acid: water (6: 4: 4: 4: 3.5, v/v/v/v/v) 5: 5: 4: 4: 3.5, v/v/v/v/v) 5: 4.5: 4.2: 3.5: 3.5, v/v/v/v/v) Run length = 65mm	Very high Rf values, diffused spots	Not satisfactory
6	Acetone: chloroform: n-butanol: glacial acetic acid: water (5: 5: 4.0: 3.5: 2.0, v/v/v/v/v) Run length = 60 mm	Good resolution, compact spots.	Satisfactory

Initial conc. (ng/spot) (A)	Quantity of std. Added (ng/spot)(B)	Total Amount (A + B)	accuracy
Initial conc. (ng/spot) (A)	Quantity of std. Added (ng/spot)(B)	Total Amount (A + B)	Peak area Mean ± S.D.	%Recovery Mean ± S.D
300	240	540	4505.5 ± 59.19	98.02 ± 0.31
300	300	600	5102.35 ±82.94	99.66 ± 2.3
300	360	660	5413.05 ±62.83	98.51± 0.54

Initial conc. (µg/spot) (A)	Quantity of std. Added (µg/spot) (B)	Total Amount (A + B)	accuracy
Initial conc. (µg/spot) (A)	Quantity of std. Added (µg/spot) (B)	Total Amount (A + B)	Peak area Mean ± S.D.	%Recovery Mean ± S.D
300	240	540	3770.8 ±78.84	99.25 ± 0.70
300	300	600	4000.35±52.51	101.9 ± 0.42
300	360	660	4103.8 ± 90.88	98.4 ± 0.28

Sr. No.	Concentration (ng/spot)	Peak Area		Rf
Sr. No.	Concentration (ng/spot)	Mean ± SD	%RSD	Rf
1	400	3343.06 ± 98.39	2.7	0.26
2	500	4183.43 ± 165.38	4.2	0.26
3	600	5001.66 ± 60.83	1.3	0.25