A Review on Analytical Methods of Diethylcarbamazine Citrate and Levocetirizine Dihydrochloride

Diethylcarbamazine citrate is an antifilarial agent widely used in the treatment of lymphatic filariasis. It acts by altering the surface structure of microfilariae, making them more susceptible to immune attack. Levocetirizine dihydrochloride is a second- generation antihistamine that is commonly prescribed for allergic conditions such as allergic rhinitis and urticaria. It works by selectively blocking peripheral histamine H1 receptors, thereby reducing allergic symptoms.

2. History

Diethylcarbamazine (DEC) citrate was first synthesized in 1947 by scientists at the American cyanamide company for the treatment of filarial infections. The development of levocetirizine is related to earlier antihistamine like hydroxyzine and cetirizine. Cetirizine, introduced 1987, contains to enantiomers, but the R-enantiomer (Levocetirizine) was found to provide they main antihistamine. Levocetirizine was later developed as a more selective and effective second-generation antihistamine with fewer side effects.

3.  Drug Profile

3.1. Diethylcarbamazine Citrate

A) Molecular structure:

B)  Molecular formula: C10 H21 N3 O

C)  Molecular weight: 391.4 g/mol

D) IUPAC: Diethylcarbamazine citrate is N, N- diethyl -4- methyl piperazine -1- carboxamide dihydrogen citrate.

E) Category:

Anti-helminthic, Anti-filarial

F) Physical property:

1. Appearance

Diethylcarbamazine is a white or almost white crystalline powder. It is odorless and possesses a slightly bitter taste. The crystalline nature of the drug facilitates easy formulation into tablets and medicated salt preparations. Because of its stability and uniform crystalline structure, it is suitable for large-scale pharmaceutical manufacturing.

2. Solubility

The drug is highly soluble in water due to its polar, ionic nature. This drug is insoluble in organic solvents like chloroform, ether and acetone. This high aqueous solubility contributes to better dissolution and helps in achieving good bioavailability when administered orally.

3. Melting point

According to thermal analysis, the melting temperature of the drug ranges from 137.90C to 144.30 C, with  peak melting  point  observed at above 140.50 C.

G) Chemical property:

1. Chemical class

Diethyl carbamazepine belongs to the piperazine group of anti-filarial components. This class of drugs he has known for its activity against helminths and parasitic worms. The piperazine Nucleus plays a significant role in altering their neuromuscular activity of parasites.

2. Stability

The drug is Chemically stable under normal storage conditions and shows good self- life. It is still stable in tablet dosage form and in medicated salt preparations used in community control programs. The stability of the drug makes it suitable for tropical climates where lymphatic filariasis is endemic.

3. Salt form

The commonly used pharmaceutical form is diethylcarbamazine citrate. The citrate salt enhances solubility, improves oral absorption, under ensures better bioavailability compared to the base form.

H) Mechanism of action:

Diethylcarbamazine primarily acts against microfilaria. The drug alters the surface membrane of the parasites, making them more susceptible to immune system attack. It enhances the host immune response by promoting adherence of leukocytes and platelets to the parasites. This immune-mediated destruction leads to rapid clearance of microfilariae from the bloodstream. Additionally, the drug may affect arachidonic acid metabolism in parasites and impair their motility. Reduction in worm movement contributes to their removal from circulation and lymphatic tissues. The exact mechanism of action of diethylcarbamazine is not fully understood. However, it is believed to act by enhancing the host immune response against filarial parasites. Studies suggest that the drug may involve platelet-mediated activity and the release of inflammatory reactions that affect the survival of microfilaria and adult worms.

Fig: 1 Mechanism of action DEC

1. Pharmacokinetics of diethylcarbamazine:

1.  Absorption

Diethylcarbamazine is rapidly and almost completely absorbed from the gastrointestinal tract following oral administration. Peak plasma concentration is generally achieved within 1to 2 hours. Rapid absorption contributes to its quick onset of action in reducing circulating microfilaria.

2. Distribution

The drug is widely distributed throughout the body tissue, including lymphatic tissue and blood, where the parasites are present. Adequate tissue penetration allows the drug to reach microfilaria and adult worms.

3. Metabolism

The drug undergoes hepatic metabolism, forming several metabolites. Some of these metabolites may also contribute to the antifilarial activity. The metabolic pathway is influenced by individual patient variability and physiological conditions.

 4. Elimination Diethylcarbamazine is primarily excreted through the kidney in urine. The elimination of half-life varies depending on urinary PH. In acetic urine, excretion is faster, while alkaline urine may prolong the half-life of the drug.

10. Pharmacodynamic of diethylcarbamazine:

The pharmacodynamic effects of diethylcarbamazine include a rapidly decreased in circulating microfilaria and suppression of parasite transmission. The drug also reduces the intensity of infection in endemic population. Clinical benefits include reduction of lymphatic inflammation, prevention of disease progression, and improvement in symptoms. The therapeutic response is influenced by parasite load, host immunity, and duration of treatment.

K) Adverse Effect:

Skin rash, Headache, Joint pain, Dizziness

L) Contraindication:

 Diethylcarbamazine is contraindicated in patients with known hypersensitivity to the drug. Patients with heavy onchocerca infection may develop severe reactions, and therefore the drug should be avoided or used carefully. Use during pregnancy requires careful risk- benefit assessment. Patients with severe neurological complications should also be monitored closely.

M) Therapeutic uses:

Diethylcarbamazine is widely used in the treatment and control of lymphatic filariasis caused by Wuchereria bancrofti and Brugia species.  It is also effective in the treatment tropical pulmonary eosinophilia.

3.2. Levocetirizine Dihydrochloride

A) Molecular structure:

B) Molecular formula: C2, H25 N2 O3 Cl, 2 HCl

C) Molecular weight: 461.8

D) IUPAC: Levocetirizine hydrochloride is (R) -2-[4-[(4-chloro-phenyl) phenylmethyl piperazin-1-y-1] ethoxy] acetic acid dihydro-chloride.

E) Category: Antihistaminic.

F) Physical properties:

1. Appearance

Levocetirizine dihydrochloride appears as a white to off-white crystalline powder.        It is odorless and exhibits a stable solid-state form under normal storage conditions.

2. Physical state

The drug exists as a solid crystalline compound at room temperature. It does not exhibit polymorphic instability under standard pharmaceutical storage conditions.

3. Melting point

The melting point ranges between 2150C – 2200C, indicating good thermal stability. It shows decomposition during melting. Meting point is used for purity identification in analysis.

4. Solubility

Levocetirizine dihydrochloride is highly soluble in water, freely soluble in methanol, and slightly soluble in ethanol. Its dihydrochloride salt from increases its solubility in polar solvent, which helps in better dissolution and improves oral absorption.

G) Chemical properties:

1. pka

Levocetirizine dihydrochloride has three ionizable groups with approximate pka values of 2.2, 2.9, and 8.0. These ionizable groups influence their solubility and degree of ionization at different pH levels, thereby affecting their absorption and pharmacological activity.

2. Partition coefficient

Levocetirizine exhibits moderate lipophilicity with a log P value around 2.0-2.2, supporting adequate membrane permeability while limiting central nervous system penetration.

3. Hygroscopic nature

The component is slightly hygroscopic and should be stored in well-closed containers protected from moisture.

H) Mechanism of action:

Levocetirizine exerts its pharmacological action through selective and high-affinity antagonism of peripheral histamine H1 receptor, which belongs to the G-protein coupled receptor (GPCR) family. Unlike simple competitive antagonists, levocetirizine functions primarily as an inverse agonist, stabilizing the inactive conformation of the H1 receptor and thereby reducing its constitutive activity. Upon allergen exposure, histamine released from mast cell bind to H1 receptors located on vascular endothelial cells, smooth muscle cells, and sensory nerve ending. Activation of H1 receptors stimulates the Gq/11 protein- mediated signaling pathway, leading to activation of phospholipase C (PLC). This results in increased intracellular calcium levels via inositol triphosphate (IP3) formation and diacylglycerol (DAG)

Fig: 2 mechanisms of action LEVC

activation, and ultimately causing vasodilation, increased vascular permeability, bronchoconstriction, and sensory nerve stimulation (itching and sneezing). Levocetirizine blocks this signaling cascade by preventing histamine binding and inhibiting downstream intercellular calcium mobilization. Additionally, it demonstrates anti- inflammatory effects independent of simple receptor blockade. It reduces:

• Eosinophil chemotaxis
• Expression of adhesion molecules such as VCAM-1
• Release of pro-inflammatory cytokines
• Late-phase allergic inflammatory response

Due to its limited penetration across the blood-brain barrier, levocetirizine shows minimal central H1 receptor occupancy, resulting in reduced sedation compared to first- generation antihistamines.

I) Pharmacokinetics of levocetirizine:

1. Absorption

Levocetirizine is rapidly and almost completely absorbed after oral administration. It exhibits nearly 100% bioavailability, indicating minimal first-pass metabolism.  Food does not significantly reduce the extent of absorption, although it may slightly delay the rate. The rapid onset of absorption contributes to first-generation antihistamines.

2. Distribution

After absorption, levocetirizine is widely distributed in the body with an apparent volume of distribution of approximately 0.4L/Kg. It is highly bound to plasma proteins (around 90%), mainly to albumin. Due to its limited ability to cross the blood-brain barrier, central nervous system penetration is minimal.

3. Metabolism

Levocetirizine undergoes minimal hepatic metabolism. It is not extensively metabolized by cytochrome P450 enzymes, which significantly reduces the risk of drug-drug interaction. Only a small fraction of the administrated dose is converted into inactive metabolites. The limited involvement of the hepatic enzymes contributes to its predictable pharmacokinetic profile.

4. Excretion

The primary route of elimination of levocetirizine is renal excretion. Approximately 85% of the administered dose is excreted unchanged in the urine. The elimination of half-life ranges between 7 to 10 hours in healthy adults. Because the drug is mainly cleared through the kidneys, dose adjustment is required in patients with renal impairment to prevent drug accumulation and toxicity.

10. Pharmacodynamic of levocetirizine:

Levocetirizine is a selective peripheral H1-histamine receptor antagonist that acts as an inverse agonist. It blocks histamine-mediated allergic responses by inhibiting vasodilation, increased vascular permeability, and itching. The drug also exhibits mild anti-inflammatory effects by reducing eosinophil activity. Due to minimal penetration into the central nervous system, it produces less sedation while providing sustained 24-hour anti-allergic action.

K) Adverse effect:

Dry mouth, Headache, Drowsiness

L) Therapeutic uses:

                                   Allergic rhinitis, Chronic idiopathic urticaria, Atopic dermatitis

M) Contraindication:

                       Hypersensitivity reactions, Severe renal impairment, Pediatric contraindication

N) Toxicology of levocetirizine:

                                        Acute toxicity, Repeat-dose toxicity, Genetic toxicology

4. Analytical Development Method

Selection of wavelength

The wavelength of diethylcarbamazine is 210 nm and levocetirizine is 231 nm. The detection wavelength of 253 nm was chosen because both diethylcarbamazine and levocetirizine strong uv absorption at this wavelength, enabling their simultaneous estimation. Selecting this wavelength ensures high sensitivity, minimal interference, and reproducible results, which is critical for the accuracy and reliability of the RP-HPLC method.

Reverse phase high performance liquid chromatography (RP-HPLC)

Reverse phase high performance liquid chromatography (RP-HPLC) in the presence study a simple and sensitive RP-HPLC method was developed and validated for the simultaneous estimation of diethylcarbamazine (DEC) and levocetirizine (LEVC) in bulk drug and tablet dosage form. The method development mainly focused on achieving good acceptable retention time and symmetric peak shapes under common chromatographic conditions.                       

Fig:3 Standard Chromatogram of DEC & LEVC

Fig:4 Sample Chromatogram of DEC & LEVC

Chromatographic separation was achieved using a Hypersil-BDS C18 column (250 ×4.6 mm, 5 µm). which is commonly used in RP-HPLC due to its good efficiency and stability. The mobile phase consisted of potassium dihydrogen orthophosphate buffer (pH 5.0) and acetonitrile.

1.  Chromatographic method

1. Selection of column

Column selection plays a major part in achieving good separation and peak shape. In this system, a Hypersil-BDS c18 column (250 ×4.6mm, 5µ) was named. C18 their non-polar nature, which provides good retention for relatively polar drug like DEC and LEVC. The named column offered better resolution, symmetric peaks, and respectable retention time. Making it suitable for routine analysis.

2. Selection of mobile phase

The mobile phase must effectively separate the analytes with minimum trailing and good resolution. Various mobile phase combinations were tried, and eventually admixture of potassium dihydrogen orthophosphate buffer (PH5.0) and acetonitrile in the ratio of 2:80 v/v was selected. The high proportion of acetonitrile helped in reducing retention time, while the buffer maintained a stable PH. This combination provided sharp, well-resolved peaks for both DEC and LEVC under isocratic conditions.

3. Selection of stationary phase

The stationary phase used in this RP-HPLC method was C18 bonded silica, which is hydrophobic in nature. The stationary phase interacts with the non-polar region of the drug molecules allowing separation based on hydrophobic interactions. Since both DEC and LEVC are moderate polar compounds, the C18 stationary phase provided efficient separation and good peak symmetry, making it ideal for simultaneous estimation.

4. Solubility of drugs

Solubility is an important factor in sample preparation and method development both diethylcarbamazine citrate and levocetirizine dihydrochloride are polar compounds and are freely soluble in aqueous medium and organic solvents like methanol and acetonitrile. In this method, the drug was found to be soluble in the mobile phase, which ensured uniform sample preparation and accurate quantification without precipitation issues.

5. Melting point of drugs

Melting point is a physical property that indicates the purity and thermal stability of a drug substance. Diethylcarbamazine citrate and levocetirizine hydrochloride possess well defined melting point, confirming their crystalline nature and purity. Although melting point is not directly involved in RP-HPLC analysis, it provides supportive pre-formulation information useful during analytical method development and drug characterization.

2.  Spectroscopy method

1. Selection of wavelength

The selection of wavelength is an important step in RP-HPLC method development because it directly affects the sensitivity and accuracy of detection. In the reported method, both diethylcarbamazine (DEC) and levocetirizine (LEVC) show adequate UV absorption in the UV region. After scanning both drugs using a UV spectrophotometer, 253nm was selected as the detection wavelength.

2. Usage of UV detector

In the present RP-HPLC method, a uv detector was used for quantification as both drugs absorb in the uv region detection was carried out at 253nm, which provided good sensitivity and well-defined peaks. Hence, RP-HPLC with uv detection was found suitable for routine analysis.

5. Method Validation

The development RP-HPLC method for the simultaneous estimation of diethylcarbamazine and levocetirizine was validated according to ICH guidelines.

1. Linearity:

Linearity of the proposed method was assessed by preparing standard solutions at different concentration levels. The calibration curves were constructed by plotting peak area versus concentration for both drugs. Linearity was established by plotting peak area versus concentration. A good linear relationship was obtained with R2 value of 0.999 for both drugs. For diethylcarbamazine, the linearity range was found between 20to 120µg/ml, while                                            

Fig:5 Linearity Data For DEC

Fig:6 Linearity Data For LEVC