Analytical method development and validation for determination of cyclizine by Gas Chromatography

Antihistamines are a group of pharmacological agents that produce their action by altering histamine levels in the H1-receptors, and in such a way, that they reverse muscle contractility in the smooth muscle, inhibiting or preventing the effects of allergic rhinitis as well as preventing other forms of hypersensitivity reaction but also to treat motion sickness, nausea, vomiting and vertigo. Additionally, due to the tranquilizing nature of antihistamines as one of its adverse effects, some of the members of this group are used in the treatment of insomnia. Moreover, certain type of antihistamines are applied in controlling neuropsychiatric and affective conditions to alleviate anxiety and cause preoperative relaxation.¹

Cyclizine, chemically is 1-benzhydryl-4-methylpiperazine having an N-alkylpiperazine in which one nitrogen of the piperazine ring is substituted by a methyl group, while the other is substituted by a diphenylmethyl group. It acts as antiemetic, cholinergic antagonist, a central nervous system, a local anaesthetic and an H1-receptor antagonist. Cyclizine a derivative of piperazine antihistamine is used as an antivertigo/antiemetic. It is used in the treatment and prevention of nausea, vomiting and dizziness that are related to motion sickness, and has also been applied in the treatment of vertigo in disorders related to the vestibular apparatus. Although the exact mechanism through which cyclizine works as an antiemetic and antivertigo agent has not been completely understood, its central anticholinergic action contributes partly to it. The medication reduces labyrinthine excitability and stimulation of the vestibular and possibly the medullary chemoreceptor trigger zone. It is also anticholinergic, antihistaminic, central nervous system depressant and local anaesthetic. Vomiting (emesis) is basically a defense mechanism of removal of irritants or otherwise unfriendly substances in the upper gastrointestinal tract.^2,3 The vomiting centre in the medulla controls emesis and is an important part that contains the chemoreceptor trigger zone (CTZ). The vomiting centre is rich in neurons that have muscarinic cholinergic and histaminergic synapses. These groups of neurons are especially engaged in conducting the signals of the vomiting centre as produced by the vestibular apparatus. The biggest problem with motion sickness is the excessive stimulation of these pathways through a range of sensory stimuli. The mechanism of action of cyclizine therefore is that of antagonism of histamine receptors at the vomiting centre therefore inhibiting the activity at this pathway. Moreover, cyclizine is also an anticholinergic, hence, it also blocks muscarinic receptors.⁴The determination of cyclizine in various drug Formulations and/or biological samples was addressed in several reports. Analytical methodology in these reports involved the use of spectrophotometry, liquid chromatograph –tandem mass spectrometry (LC/MS/MS), high performance liquid chromatography (HPLC) with fluorescence detection, Capillary Zone Electrophoresis (CZE), whereas no gas chromatography approach has been performed to this drug.^5-7 This work describes a new simple, direct and selective capillary GC/FID method for the analysis of cyclizine.

Materials-

Reference standard of cyclizine, analytical grade Acetonitrile (Merck Specialities Pvt. Ltd., Mumbai, India), 0.45 µm Millipore syringe filters (Ultipor^®N₆₆^®Nylon Membrane) were given by Aadhaar Life Sciences, Solapur, Maharashtra, India. The tablet sample brand name used in analysis was Valoid 50 mg.

Instrument-

Agilent 7890B gas chromatography (GC) system equipped with a split injector and a flame ionization detector (FID) was used in this study. Nitrogen (ultrapure) was obtained from G 1010E nitrogen generator (Peak scientific) and used as carrier gas. 5μL gas-tight syringe (SGE Analytical science) were used.

Standard Solutions preparation-

For the preparation of Cyclizine Standard Solution (CSS), a precise 10 mg of cyclizine was accurately weighed and transferred into a volumetric flask along with the addition of 1 ml 1N sodium hydroxide and this mixture is dissolved in 5ml of acetonitrile and final volume made up to 10 ml (1000µg/ml). This standard solution was used for the analytical method development.

Drug product solution preparation-

For internal standard solution, 10 tablets were taken and were weighed to determine average tablet weight. Tablets were crushed in mortar & pestle, tablet powder equivalent to 10 mg of drug was weighed in 10 ml volumetric flask and 1 ml of 1N Sodium hydroxide was added to it and diluent was added upto the mark and sonicated for 5 minutes. Later the solution was filtered through 0.45-micron membrane filter (Label claim: Each tablet contained 50 mg of Cyclizine).

RESULTS AND DISCUSSION-

Chromatography-

A very specific and sensitive capillary column gas chromatographic technique was designed to determine cyclizine and use HP-5 column (30mm length x0.32mm internal diameter x 0.25mm film thickness). A 0.5µL standard solution was injected through the autosampler in the analysis; the sample injected was sent to a detector at 300°C with a ratio of 50:1 split. Symmetrical sharp peak was achieved by isothermal programming inlet temperature at 280°C. The elution time of cyclizine was 5.48min. The figure (2) shows the chromatogram of drug product and figure (3) shows the chromatogram of working standard for cyclizine.

Figure 2: Typical GC-FID chromatogram for drug product of cyclizine

Figure 3: Typical GC-FID chromatogram for working standard of cyclizine

Linearity-

Linearity of an analytical procedure refers to the ability of an analytical procedure, over a given range, to give test results that are proportional to the concentration (amount) of the analyte in the sample.⁸ Relationship between concentration of analyte and relative peak area was later explored. The linear equation obtained by the least squares method for cyclizine was found to be y = 843.17x - 1888.1 as shown in (Fig. 4). The resulting correlation coefficients allowing estimating the quality of the curve was R² = 0.9997, these values of R² greater than 0.99 indicates a satisfactory linearity. The linearity was demonstrated in the concentration range (600-1400µg/mL).

Figure 4: Linearity line of cyclizine

Accuracy-

Accuracy of an analytical procedure is the expression of the proximity of assent of the observed value accepted as a conventional true value or accepted reference value and the measured value.^9,10 In the case of a quantitative method, at least nine values should be determined over the spectrum of values specified.

Table 1-Results of accuracy by proposed GC method

Accuracy was determined from three concentrations of cyclizine, with six replicates. Results are reported in (Table1). For cyclizine, the results indicated that the individual recovery ranged from 99.81% to 99.16%. Considering the same criteria, the recovery of cyclizine by the proposed method was also satisfactory, with the RSD of 0.13%.

Assay-

An assay is a validated analytical test that measures the concentration or strength of an analyte in a pharmaceutical substance, formulation, or biological sample.^9,10 In concern with the assay measurement, working standard and drug product are evaluated along with the blank solution. The recovery of cyclizine by the proposed method was acceptable, as the mean % recovery value of assay of cyclizine was found to be 99.42%.

Precision-

Accuracy of an analytical process refers to the level of consistency of a set of measurements made by various samplings of a single homogenous sample under given conditions. Intra-day precision is the consistency of the method on the same day, under the same operating conditions, and inter-day precision is the consistency of the method manufacturing that is conducted on different days.¹⁰ The solution was injected six times (N = 6). The results of intraday precision (Repeatability) are reported in (Table 2). The % average assay of intra-day precision and % RSD was found to be 99.36% and 0.18 and whereas % average assay of inter-day precision and % RSD was found to be 98.19% and 0.69. The intra-day and inter-day precision of the method were evaluated and the %RSD values were found to be less than 2%, indicating that the method is precise.

Table 2- Results for repeatability of proposed GC method