INTRODUCTION:

Mannitol, an osmotic diuretic derived from naturally occurring alcohols in plants and fruits, is one such substance. mannitol was discovered in 1806 by Joseph Louis Proust. This is a pleasant, white, odourless powder or free-flowing granules¹ that may be found. “An IV or oral route may be utilised for the D-enantiomer of Mannitol known as D-Mannitol ((2R, 3R, 4R)-hexane-1, 2, 3, 4, 5, 6-hexol).”².

Figure 1: Structure of mannitol

Diuresis is elicited by mannitol. Because of its ability to promote dehydration, mannitol is also used in neurosurgery and neurology³. Ocular pressure should be reduced by using Mannitol⁴. Because it is eliminated unaltered via the kidneys, impaired renal function results in cellular dehydration⁵. Additionally, mannitol has been shown to temporarily break the blood-brain barrier, enabling chemotherapeutic medicines to enter the brain more easily. Because of its superior portrayal, greater availability, and cheaper cost, mannitol is the most often utilised intestinal contrast in magnetic resonance enterography⁶. With an acceptable safety profile, mannitol has been clinically shown to improve airway function in cystic fibrosis⁷. IDPM bronchial provocation test to assess bronchial reactivity in asthma⁸.

By 2024, it is expected that the mannitol market would grow to USD 418.3 million⁹. In most nations, mannitol is a vital drug. One of the few sugars that has no or little influence on blood glucose levels is Mannitol. The low-calorie content (1.6 cal/g) is an additional benefit for diabetics. Ten to ninety percent (w/w) of tablet formulations include mannitol as an excipient diluent. Non-toxic, water-soluble, non-hygroscopic, and only moderately heat resistant. In general, this is regarded as safe and widely used in the form of solid dosages.¹⁰

It reduces intracranial pressure by improving the tonicity of blood plasma after intravenous administration. Mannitol cannot cross the blood-brain barrier, but its enhanced tonicity causes the brain's parenchyma to release water, which lowers intracranial pressure. As mannitol causes the kidneys to expel this water in the form of urine, it contributes to moderate dehydration of brain cells. By increasing blood plasma tonicity, mannitol moves water from the eye's vitreous humour into the intravascular space, which lowers intraocular pressure and protects the retina. Oliguria-induced fluid overload necessitated the administration of mannitol and fluid to boost urine production. Nonetheless, the use of mannitol in these circumstances is out of date since it might lead to acute tubular necrosis¹¹. Water and other toxins are drawn out of blood during excretion, lowering their concentration in the blood¹².

Analytical methods are important component in drug development.^13,14 According to the findings of the literature study, multiple studies have been conducted to determine the amount of Mannitol present in different matrixes. Various available methods are Spectrophotometry, chromatography, and few analytical methods. The details and review of published analytical method is already published in separate publication¹⁵. Authors suggest readers to refer that paper in this regard. But literature also reveals that the acid-dye method has been developed till date. The presented study is a simple, economical, and fast method for the analyst of Mannitol I.V formulation. The method was validated by the ICH guideline.

MATERIALS AND METHODS:

Mannitol was procured from Central drug House (P) ltd, chloroform, HCl and methylene blue from Merck. Freshly prepared double distilled water using inhouse distillation assembly was used in this study. The Shimadzu 1800 Double Beam - UV Vis spectrophotometer with pair of 10 mm matched quartz cell, Shimadzu - AX220 Digital Balance and Labman pH meter (Model LMPH-10) was used in this study.

Method Development Study

Preparation of 0.1N HCl

Take 8.5 ml of concentrated HCL and transfer it into 1000 ml of volumetric and volume makeup with distilled water and sonicate it for 10 min.

Preparation of Dye

Take 100 mg of methylene blue and transfer it into 100ml volumetric and volume makeup with distilled water and sonicate it for 10 min. (stock solution). Pipette out 10 mL of stock solution and place it into a 100mL volumetric flask, then top it with distilled water to get the desired volume.

Optimization of the reaction conditions

The reaction was tuned using two parameters, namely, the volume of acid in the solution, the volume of dye and concentration, and the amount of time the solution was shaken (in minutes). To ensure the greatest stability of the chromophore, 0.1 µg/mL 5 ml of dye and 10 ml of 0.1N HCl were used, with the total volume and shaking duration being 2 minutes.

Shaking time

As the medication was soluble in HCl and water and colour in water thus. Therefore, the shake duration should be substantial enough medicine and dye become blended in 2 min shaking period. A two-minute interval of shaking was selected for the extraction procedure to ensure that the ion-pair of drug and dye was successfully transferred from the liquid to the solid phases of the solution. Using chloroform extraction, it was discovered that the drug was soluble in both water and dye, and this resulted in the creation of an ion-pair in an aqueous layer. Add 5ml of dye (methylene blue) to the sample in the conical flask and stir it for 5 min. Take 10 separating funnels and add 10ml of chloroform into them and pour the sample with dye into it and shake them all for 2 min. Aqueous layer was removed using separating funnel and was used for measuring absorbance. The wavelength selected for study was 653 nm.

Method Validation

Linearity

In the field of analytical chemistry, linear processes are defined as processes that provide test findings that are directly proportional to the concentration of analyte in each sample. We employed successive dilutions of the stock solution of D-Mannitol as a test for the linearity of the detector response. D-mannitol was produced and examined at ten different concentrations, including 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 µg/ml (Figure 2). The calibration curve is shown in Figure 3.

Figure 2: Overlay graph of Standard Curve of Mannitol (4, 5, 6, 7, 8, 9, 10, 11, 12, 13 µg/ml)

Figure 3: Calibration curve of proposed method

Precision

Repeatability

The dilution of 4 µg/ml was prepared, and relative standard deviation was calculated after six consecutive readings using UV spectrophotometer. The % RSD found was 0.359 shows method is repeatable.

Interday

The interday precision was calculated by using three replicates of 4, 8 and 12 µg/ml at three different times in a single day. The % RSD found was below 1% at each concentration level shows method qualifies this criterion also.

Intraday

The percentage RSD was calculated in the same way as mentioned under interday precision in three consecutive days. The %RSD was below 1% at each level again shows the method is precise to be used further.

Accuracy

The dilutions 6, 8, and 10 µg/mL of the injectable solution of marketed preparations of mannitol were prepared and analyzed by using the proposed method.

Table 1: Result of Accuracy

S.No	Conc. µg/mL (n=3)	Result	Recovery %
1.	6	5.98	99.66
		5.93	98.33
		6.1	101.6
2.	8	8.01	100.1
		7.99	99.8
		8.0	100
3.	10	9.99	99.9
		10.1	101
		9.98	99.8

The trueness studies shown that percentage recovery is between 98.33 to 101.6 % shows method is accurate.

LOD and LOQ

The limit of detection and limit of quantitation was calculated as per given formula.

LOD=3Sa /b,

LOQ=10Sa /b,

Where Sa is the standard deviation of the response and b is the slope of the calibration curve.[16] The LOD and LOQ values found were 0.031 and 0.093 µg/ml.

Application of the proposed method

The mannitol injection procured from the market contains 20 % w/v (labelled amount). The dilution was prepared by taking 1 ml of solution in 100 ml water. The prepared solution was further diluted in distilled water to prepare target concentration. The standard solution was also prepared using pure Mannitol as per method already described in the material and method section. The analysis was performed by comparing both results. The concentration of drug in marketed injectable formulation was found to be 99.9%.

CONCLUSION:

Mannitol is a medicine that is also used as an excipient in a variety of pharmaceutical formulations. It may be used as a sugar substitute for diabetics because of its sweet flavour, which means it is poorly absorbed from the colon. Mannitol measurement in diverse matrices has been accomplished using a variety of spectrophotometry and chromatographic methods that have been developed and verified. Tables 4 and 5 show the spectrophotometry and chromatography procedures, respectively. Mannitol is an aliphatic molecule with no aromatic rings that is difficult to test using UV spectrophotometry. As previously stated, most of the spectrophotometry processes that have been devised are predicated on the enzymatic conversion of mannitol into a form that is adequate for effective observations. These techniques are time-consuming and labor-intensive, and they need the growth of additional enzymes. Other chemical processes may be used to determine mannitol, such as coupling it with any dye using simple ionic interactions, as in acid dye procedures, or adding an aromatic component, such as benzene, to its structure, which allows it to be detected using UV spectrophotometers. Even while other chromatographic technologies, such as high-performance liquid chromatography (HPLC), make use of an aqueous mobile phase, these procedures are expensive, time-consuming, and require the use of a particular kind of column. Furthermore, a few described techniques include non-aqueous phases in mobile phases in some percentage.

The developed method is simple UV spectrophotometric determination of Mannitol successfully utilized in injectable formulations. The validation parameters were specificity, linearity, precision, accuracy, limit of detection and limit of quantitation. The linearity studies show that developed method can be used for determination of Mannitol from 4 to 13 µg/ml. The specificity studies are not required in the proposed method since the injection in water only. The validation studies shown all results within specified range; thus, the developed method is specific, precise and accurate for determination of Mannitol in parenteral formulations. The details of validation studies are given under Table 2.

Table 2: Summary of results of validation studies

S No	Validation parameter	Observations
1	Specificity	Not required
2	Linearity	4-13 µg/ml
3	Precision Repeatibility Interdaay Intraday	0.58656 0.56533 0.022189
4	Accuracy	98.33 to 101.6
5	Limit of detection (LOD)	0.031
6	Limit of Quantitation (LOQ)	0.093

CONFLICT OF INTEREST:

None declared.

ACKNOWLEDGEMENT:

Authors acknowledge the support given by KIET Group of Institutions (KIET School of Pharmacy) for providing necessary facilities.

SOURCE OF FUNDING:

KIET group of Institutions

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Received on 04.11.2022 Modified on 26.07.2023

Research J. Pharm. and Tech 2024; 17(7):3177-3180.

DOI: 10.52711/0974-360X.2024.00496