A green analytical method for the determination of glucosamine using FTIR spectrophotometry

Glucosamine is an amino sugar, naturally found in cartilage in between the joints. Glucosamine is prescribed for the treatment of osteoarthritis as it may either increase the content of cartilage or inhibit the enzyme responsible for the degradation of cartilage. As per the literature survey, glucosamine was analyzed using different methods, such as High performance liquid chromatography (HPLC) and UV Spectrophotometry, but none was carried out using Fourier Transform Infrared (FTIR) Spectroscopy. Therefore, the present work aims to carry out a green method for the analysis of glucosamine in marketed tablets using FTIR. The qualitative and quantitative analysis of glucosamine was carried out using the KBr pellet method in the mid-infrared region. The method involves the direct measurement of absorbance of the amine group peak at 3309 and 3356 cm−1. The proposed method is validated according to the ICH guidelines (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Excellent linearity was obtained in the concentration range of 2–7%w/w (R2 = 0.9902). The percentage purity of the glucosamine tablet was found to be 99.629%. The results obtained indicate that the proposed method used to analyze glucosamine is precise, accurate, and reproducible. Thus, a simple, rapid, economical, and environmentfriendly method was developed and validated for the analysis of glucosamine in marketed tablets and it can be used for the quality control of routine analysis of this drug.


INTRODUCTION
Glucosamine is an amino monosaccharide ( Figure 1) (Chen et al., 2006) and is a natural component of cartilage which produces varieties of chemicals involved in the building of tendons, ligament, cartilage, and fluid surrounding the joints (Kirkham and Samarasinghe, 2009). The cartilage breaks down in an osteoarthritis patient (Hag et al., 2003). The glucosamine supplement may either increase the content of the cartilage or inhibit the enzyme destroying the cartilage (Papich, 2016). Therefore, it is a popular over-the-counter nutraceutical supplement frequently combined with chondroitin for the treatment of osteoarthritis and other joint pain (Dasgupta and Klein, 2014). Oral administration of glucosamine has been reported to show the clinical effect of re-establishing the lost cartilage and slowing down the joint deterioration without significant adverse effects (Reginster et al., 2012).
There are several published research studies on the development of an analytical method for the analysis of glucosamine using the HPLC method (Hadad et al., 2012;Harmita et al., 2017;Shao et al., 2004). However, glucosamine lacks chromophore and hence it is a prerequisite to derivatized glucosamine using a suitable derivatizing agent to impart UV-absorbing chromophore so that it can be detected in the UV region (Zhou et al., 2004). As per the literature survey, there is no research carried out for the estimation of glucosamine by infrared spectroscopy. The use of IR spectroscopy for quantitative analysis has several advantages compared to other analytical methods such as HPLC which require the toxic solvent for the operator as well as for the environment (Fahelelbom et al., 2020). The infrared spectroscopy method stands out when there is a need for the replacement of a method that requires the use of a large amount of organic solvents compared to the method that does not use them. This method allows the quantification of substances without the use of any solvent, particularly organic solvents, which makes the method environment-friendly by following the principles of green chemistry.
This work describes the simple and environment-friendly infrared spectroscopy method as a green tool for the quantification of glucosamine in marketed tablets.

Instrumentations
The estimation of glucosamine was carried out in a Shimadzu FTIR connected to an IR solution software. A mechanical pellet press machine equipped with a pressure gauge was used to create the thin transparent glucosamine pellets. Other types of equipment used are analytical weighing balance and mortar and pestle. Table 1 presents the FTIR conditions used for the analysis.

Chemical and reagents
Standard glucosamine HCl and glucosamine sulfate potassium chloride tablets 750 mg (Jointace) were purchased from a pharmacy shop. Potassium bromide (KBr) IR grade was also used.

Qualitative analysis
The qualitative analysis of glucosamine was carried out by the KBr method using FTIR in the mid-infrared region. 1 mg of glucosamine standard was weighed and mixed with 100 mg of IR grade KBr using a mortar and pestle. After properly mixing glucosamine and KBr (1:100), a thin translucent pellet was prepared by pressing the mixture under the mechanical pellet press at 10 tons for 2 minutes. The sample was also made in translucent pellets using the same procedure. The transmittance spectra of both standard and sample glucosamine were obtained in the IR solution software in the range of 4000-400 cm −1 in 20 scans. The spectrum of the glucosamine sample obtained was compared with that of the spectra of glucosamine standard to verify the similarity between the two specta.

Standard calibration curve
For the estimation of glucosamine in the sample, the standard calibration curve was obtained by plotting different concentrations of the standard glucosamine HCl against their absorbance intensity. The different concentrations of glucosamine ranging from 1 to 7%w/w were prepared by homogeneous mixing of glucosamine HCl with KBr. 75 mg of each mixture was pressed in a mechanical press under a pressure of 10 tons for 2 minutes to get a uniform thin translucent pellet. The spectrum of each concentration was obtained in absorbance mode in 20 scans. The absorbance intensity of each concentration at 3309 and 3356 cm −1 wavenumbers was selected which is the characteristic band of glucosamine (amine group). Table 2 shows the parameters of the standard calibration curve obtained.

Estimation of glucosamine in pharmaceutical dosage form
Ten glucosamine tablets (Jointace) were weighed and the average weight was calculated. The tablets were ground into a fine powder. Weight of powder equivalent to 7 mg of glucosamine was weighed and mix thoroughly with 93 mg of KBr. 75 mg of the homogenous mixture was made into a translucent pellet and the absorbance spectrum was measured. The amount of glucosamine in the tablet was determined using Eq. (1) and the percentage of glucosamine content was calculated from Eq. (2) as follows: where C a is the concentration of glucosamine in the sample (%w/w), A a is the absorbance intensity of the sample pellet, C b is the concentration of glucosamine in the standard, A b is the absorbance intensity of the standard pellet, and C t is the theoretical concentration of glucosamine in the sample.

Qualitative analysis
The spectrum of the glucosamine sample obtained was compared with that of the spectrum of the glucosamine standard to verify the similarity between the two spectra. Figure 2 shows an overlay spectrum of the standard glucosamine and glucosamine tablet (Jointace). It is clearly shown that the same absorption band is observed in both spectra. The similarity between the spectra is strongly indicative of the identity of glucosamine in the pharmaceutical marketed tablet.

Quantitative analysis
The absorbance of different concentrations of the standard glucosamine ranging from 2 to 7%w/w was measured at 3309 cm −1 and the overlay spectrum of the standard glucosamine in absorbance mode was established, as shown in Figure 3. The absorbance of the glucosamine tablet was also measured, as shown in Figure 4. A comparison between the absorbance at 3309 cm −1 of both reference standard and glucosamine tablet was carried out and the amount of glucosamine content and percentage purity glucosamine was calculated as shown in Table 3.

Linearity
The linearity of the standard glucosamine HCl was carried out in the concentration range of 2-7%w/w. Table 4 shows the linear concentration range and its proportional increase in absorbance. The calibration curve was constructed by plotting the concentration of glucosamine against the absorbance intensity at the 3309 and 3356 cm −1 wavenumbers and all the regression parameters were calculated. Excellent linearity was obtained as indicated by the coefficient correlation value of both wavenumbers as shown in Figures 5 and 6.

Precision
The precision of the analytical method is the degree of closeness in the measured values. The precision of the proposed method was assessed by the intra-day and inter-day precision studies. The intraday and inter-day precision studies were carried out by measuring the absorbance of glucosamine standards at a concentration 5%w/w in six replicates and the % RSD (Relative standard deviation) was calculated. The result obtained was precise as indicated by the %RSD of 0.431871 and 1.291162 for intra-day and inter-day studies, respectively, as shown in Table 5.

Accuracy
The accuracy of the method is the closeness of the measured value to the actual value. The accuracy of the method was evaluated by calculating the percentage recovery of the glucosamine by standard addition method at 50%, 100%, and 150% levels. 2, 4, and 6 mg standard glucosamine HCl were added to a known amount of the sample, and a pellet of spiked concentration was prepared and absorbance was measured in three replicates. Table 6 shows the accuracy studies where the percentage recovery at all the levels was between 98% and 100%, which indicates that the method is accurate.

Robustness
The reliability of the method was assessed by the robustness studies. The robustness is the ability of the method     to remain unaffected by small changes in the methodology. The robustness of the proposed method was carried out by small deliberate alterations in the parameters, such as time and pressure applied to create the translucent pellet. The %RSD obtained in the study was between 0.4 and 0.9 as shown in Table 7, indicating that the proposed method is reliable.

Ruggedness
The reproducibility of the test result was measured by the ruggedness studies. Ruggedness is the ability to remain uninfluenced by alteration in external factors, such as variation in the operation and environmental conditions. The reproducibility of the test result was carried out by the two different analysts and the %RSDs obtained were 0.401055 and 0.431871 as present in Table 8, indicating that the method is reproducible.

Limit of quantification and limit of detection
LOD (limit of detection) and LOQ (limit of quantification) is the lowest amount of analyte that can be detected and quantified. It is calculated from the slope of the calibration curve and the standard deviation using Equations (3) and (4) as follows:

LOD = 3.3*σ/S
(3) where σ is the standard deviation and S is the slope of the calibration curve. The LOD and LOQ of the proposed method are presented in Table 9 and the low value of both parameters indicates the high sensitivity of this method.

DISCUSSION
The analysis of glucosamine using FTIR as a green analytical tool was accomplished by an environment-friendly method. The FTIR spectrum of glucosamine in bulk and the marketed tablet for the qualitative and quantitative estimation was obtained directly by scanning the pellets obtained from the solid    powder of the glucosamine without the use of any solvent, which reduces the solubility challenges of glucosamine. Glucosamine analysis by UV spectroscopy and HPLC coupled with a normal detector, such as UV and fluorescence detector, requires the derivatization step before the analysis since glucosamine does not contain chromophores or fluorophores that enable the detection of the analyte in the UV-visible region. However, the FTIR method did not depend on the detection in the UV-visible region, eliminating the requirement of an extra derivatization step which is tedious and time-consuming. Therefore, the FTIR method is environment-friendly and economical with a short analysis time.
When the validated parameters for the method developed were evaluated, the method shows excellent linearity as indicated by the regression coefficient value obtained and the %RSD of the validated parameters are below the limit (<2%) signifying that the proposed method is precise, accurate, reproducible, and reliable. The LOQ value obtained acknowledges the high sensitivity of the method.

CONCLUSION
A green analytical method using FTIR spectroscopy in the mid-infrared region for the analysis of the glucosamine was developed and validated. The proposed method was found to be sensitive, accurate, precise, reliable, and reproducible. The novelty of the method arises from excluding the need for the use of any solvent and derivatization step which is requisite for other analytical methods, such as chromatographic and UV spectrophotometric methods, for the analysis of glucosamine. The virtue of the present work includes a simple, fast, economical, and environment-friendly method compared to the other methods. Therefore, it can be used for quality control of routine analysis.

CONFLICT OF INTEREST FUNDING
None.