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

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.

Table 1. FTIR conditions. [Click here to view]

Quantitative analysis

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:

${\mathit{C}}_{\mathrm{a}}={\mathit{A}}_{\mathrm{a}}\times {\mathit{C}}_{\mathrm{b}}/{\mathit{A}}_{\mathrm{b}}(1)$

$\%{\mathit{C}}_{\mathrm{a}}={\mathit{C}}_{\mathrm{a}}/{\mathit{c}}_{\mathrm{t}}\times 100(2)$

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.

Table 2. Standard calibration data of glucosamine HCl. [Click here to view]

Figure 2. Overlay spectrum of standard glucosamine HCl and glucosamine tablets (Jointace). [Click here to view]

Figure 3. Overlay spectrum of different concentrations of standard glucosamine HCl. [Click here to view]

Figure 4. FTIR spectra of glucosamine tablets (Jointace) in absorbance mode. [Click here to view]

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.

Method validation

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.

Table 3. Determination of glucosamine in tablet. [Click here to view]

Table 4. The linearity of the glucosamine HCl. [Click here to view]

Figure 5. Calibration curve of glucosamine absorbance at 3309 cm−1. [Click here to view]

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.

Figure 6. Calibration curve of glucosamine absorbance at 3356 cm−1. [Click here to view]

Table 5. The precision study of glucosamine HCl. [Click here to view]

Table 6. Accuracy study of glucosamine HCl. [Click here to view]

Table 7. Robustness study of glucosamine HCl. [Click here to view]

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:

$\mathrm{LOD}=3.3*\sigma /\mathrm{S}(3)$

$\mathrm{LOQ}=10*\sigma /\mathrm{S}(4)$

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.

Table 8. Ruggedness study of glucosamine HCl. [Click here to view]

Table 9. LOD and LOQ of the glucosamine HCl. [Click here to view]