TECHNIQUES FOR ANALYSIS OF GLUCOSAMINE

There are many challenges involved in the analysis of glucosamine. First, the hydrophilic nature of the molecule makes the extraction with organic solvents from plasma ineffectual. Along with the drug, a variety of endogenous compounds with a chemical structure similar to glucosamine, such as glucose, galactose, other sugars and amino sugars [Huang et al. 2006b], would also be extracted from a biological sample [Zhang et al. 2006]. To circumvent this, often lengthy sample preparation techniques are used. Second, several ingredients are often found within a commer-cial formulation of glucosamine. Chondroitin, also a glycoaminoglycan but with molecular weight of 5,000–55,000, is commonly found in such formulations, and this compound has the potential for interference with the quantitative analysis of glucosamine [Shen, Yang, and Tomellini 2007]. Analysis of chondroitin is not nearly as highly developed as that for glucosamine.

Radiolabeling

Previous analytical methods for biological samples depended on radiolabeling a compound; however, using radioactivity to quantify glucosamine may potentially confound results because the parent drug molecule cannot be differentiated from its degradation products and metabolites [Huang et al. 2006a; Zhang et al. 2006;

Shen, Yang, and Tomellini 2007]. Therefore, there is a need for specifi c analytical techniques to analyze the content of glucosamine in both commercial products and biological fl uids. Both areas present different problems in terms of analysis. A vari-ety of methods have been suggested in an attempt to overcome such challenges, with each method having its own applications, limitations, and advantages, which will be discussed below.

HPLC Separation

It has been suggested that, of all the methods, HPLC is the most extensively used and highly sensitive technique for the analysis of glucosamine [Huang et al. 2006b].

Glucosamine poses several challenges for analysis using liquid chromatographic methods, and several techniques have been used and studied. The fi rst challenges are created by the nature of the glucosamine molecule (Figure 7.1).

Because sugars and amino sugars (such as glucosamine) are highly polar mol-ecules, they are not retained on common hydrophobic HPLC column packing materials (e.g., RP C18), which makes separation diffi cult [Roda et al. 2006]. A possible solution to this problem is the use of more expensive amino columns [Shao et al. 2004] or the complexation of glucosamine through its amino group with a hydrophobic compound. The chemical structure of glucosamine also lacks a suit-able chromophore or fl uorophores, which absorbs in the wavelength range useful for HPLC with either UV or fl uorescence detection [Nemati et al. 2007; Shen, Yang, and Tomellini 2007].

Precolumn Derivatization Agents for UV/Fluorescent Visualization To overcome this problem, glucosamine is often derivatized to incorporate a suitable chromophore or fl uorophore into its structure to improve its detection by UV methods [Shen Yang, and Tomellini 2007], and such agents can be bound to glucosamine through its amino group.

It has been reported that precolumn derivatization steps can often be lengthy, making the time of analysis for such techniques unsuitable for routine use [Shen, Yang, and Tomellini 2007].

Issues with the stability of glucosa mine-derivative complexes have been raised.

Shen, Yang, and Tomellini [2007] monitored the UV absorption of a glucosamine derivative σ-phthalaldehyde-3-mercaptopropionic acid (OPA-MPA) over time to illustrate stability issues. The derivatization reaction was performed with an OPA-MPA molar ratio of 1:50, according to a published method in the U.S. Pharmacopeia.

The 1:50 ratio was shown to increase stability and was therefore the chosen method.

The maximum absorption of the glucosamine-OPA-MPA derivative in a borate buf-fer (80 mM, pH 9.5) was found to occur at 335 nm. The absorbance was measured for

4.5 h after mixing the reagents. Shen, Yang, and Tomellini [2007] showed that the UV absorbance decreased to approxi-mately half the maximum over a period of 4 h. This insta-bility could lead to inaccuracy in analysis if the time period varies between performing the derivatization reaction and injection of the sample into the HPLC system.

Several agents have been suggested as suitable to make detectable complexes with glucosamine. Studies investigated the usefulness of each agent for a variety of applications, such as glucosamine content in a commercial product, or biological

Figure 7.1 The

fl uid analysis. Possible precolumn derivatization agents include the following: OPA [Nemati et al. 2007], phenylisothiocyanate (PITC) [Liang et al. 1999; B. Lockwood 2007], N-(9-fl uorenyl-methoxycarbonyloxy) succinimide, and 9-fl uorenyl-methyl chloroformate (FMOC-Cl) [Huang et al. 2006b; Zhang et al. 2006].

All of these agents can be complexed with glucosamine precolumn before separation. Nemati et al. [2007] investigated the use of OPA-MPA as a precolumn derivatization agent for analysis of glucosamine. The derivatization reagent was made by dissolving 5 mg of OPA in 900 μl of methanol, 100 μl of borate buffers, and 10 μl of MPA solutions. Separation was achieved using a 250 × 4 mm RP C18 column. The mobile phase was isocratic and consisted of methanol-sodium phos-phate (pH 6.5, 12.5 mM; 10+90, volume/volume [v/v]) and methanol-tetrahydrofuran (97+3, v/v) for 20 min at a fl ow rate of 1 ml/min in proportions of 85+15. Detection was performed using a fl uorescence detector. A calibration curve was constructed using standards ranging from 5 to 200% of the nominal assay concentration of 10 μg/

ml glucosamine HCl, which showed excellent linearity with a correlation coeffi cient of 0.9980. The lower limit of detection (LOD) and quantifi cation (LOQ) were 0.009 and 0.027 μg/ml, respectively [Nemati et al. 2007]. Nemati et al. concluded that derivatization with OPA-MPA was extremely simple and robust and showed greater sensitivity than other derivatization methods. The derivatization reaction took less than 5 min. The process would also be suitable for automation because there is no need to remove excess derivatization agent, and the reaction does not require an evaporation step, which would reduce human error. OPA also has the advantage that both UV and fl uorescence detectors can be used, giving it a wider applica-bility [Nemati et al. 2007]. Staapplica-bility issues with OPA have been noted, because glucosamine-OPA complexes decompose over time [Huang et al. 2006a]. The analysis of glucosamine in human fl uids requires different analytical techniques and is essen-tial for understanding the physiological role of glucosamine, metabolism, pharmacoki-netics, fate, and mechanism of action. Huang et al. investigated the use of FMOC-Cl as a derivatization agent for the analysis of glucosamine content in human plasma.

The levels of glucosamine in human plasma are relatively low, which presents an additional analytical challenge. A suitable analytical technique therefore needs to be sensitive, simple, and fast. Derivatization reagent consisted of 0.8 mM FMOC-Cl in acetonitrile. Separation was achieved using a C18 analytical column, 150 × 4.6 mm inner diameter, 5 μm. Elution was obtained by using gradient steps of solvents A (acetonitrile) and B (water): 30:70 (A/B) for 10 min and then 98:2 for 15 min at a fl ow rate of 1 ml/min. The LOD was found to be 15 ng/ml (signal to noise ratio

≥3), which is well below the expected drug concentration in plasma samples from a patient given therapeutic doses of glucosamine sulphate. Huang concluded that the use of FMOC-Cl as a derivatization agent allowed for sensitive, rapid (attributable to faster sample preparation times), simple, and reliable analytical results. It has also been shown that there should be no stability issues with the FMOC-Cl-glucosamine complexes, because samples have been shown to be stable for 24 h at 4ºC [Zhang et al. 2006]. Thus, the technique should be appropriate for the routine analysis of glucosamine for pharmacokinetic, bioavailability, or bioequivalence studies [Huang et al. 2006b]

Postcolumn Agents Used for Indirect Fluorescent Detection

Another method used to overcome the issue of the limited UV or fl uorescence detection attributed to glucosamine is to attach a visualizing agent postcolumn separation. Shen, Yang, and Tomellini [2007] exploited an indirect fl uorescence detection method for the analysis of glucosamine in dietary supplements. This method claimed to avoid the stability and time issues associated with precolumn derivatization and thus could be advantageous. The method is based on the fl uorescent signal of either L-tryptophan (L-TRP) or DL-5-methoxytryptophan (5-MTP). Either compound is added postcolumn as a copper complex; when bound to the copper (II) ion, the fl uorescent signal of these compounds is quenched, thus giving no absorbance.

Glucosamine is capable of complexing with the copper (II) ion and thus displaces some fraction of the L-TRP or 5-MTP recovering their fl uorescent signal. The amount of glucosamine present can be calculated indirectly from the intensity of signal produced by the displaced L-TRP or 5-MTP. Shen, Yang, and Tomellini used the following chromatographic conditions: mobile phase, 1.6 mM sodium borate, pH 9.0; fl ow rate, 1 ml/min; postcolumn interaction component 2 × 10⁻⁵ M CU (L-TRP)₂ in 40 mM sodium borate at pH 9.0, or 2 × 10⁻⁵ M Cu (5-MTP)₂ in 40 mM sodium borate at pH 8.4; fl ow rate, 1 ml/min; column, strong anion-exchange column, PRP-X100 (250 × 4.1 mm, 10 μm) with a fl uorescence detector [Shen, Yang, and Tomellini 2007]. The main disadvantage reported was the lower sensitivity of the method compared with precolumn derivatization. The detection limit found using postcolumn derivatiza-tion corresponded to a concentraderivatiza-tion of glucosamine of 3.2 μg/ml compared with detection limits of 0.009 μg/ml [Nemati et al. 2007] and 0.075 μg/ml [Ji et al. 2005]

for precolumn derivatization with OPA-MPA and PITC, respectively. However, the technique would still be an acceptable alternative if detection limits were not an issue (e.g., in the analysis of glucosamine in commercial products) because it avoids the time-consuming derivatization reactions and avoids the possibility of stability issues [Shen, Yang, and Tomellini 2007].

HPLC Using Electrospray Ionization-MS Detection

Because of the possible limitations in sensitivity of the methods involving precolumn and postcolumn derivatization followed by UV or fl uorescence detection, researchers investigated the possibility of HPLC separation followed by MS detection [Huang et al. 2006a; Roda et al. 2006].

A method developed by Huang et al. looked at using precolumn derivatization with PITC, followed by electrospray ionization (ESI)-MS detection. Huang used an RP C18 column, and, because of the high polarity of glucosamine, the elution was too fast; thus, suitable derivatization was used to help facilitate HPLC component separation. PITC was chosen specifi cally over other agents because it gave selective mass spectra. Elution was performed with 0.2% glacial acetic acid (A) and methanol (B) at a fl ow rate of 0.3 ml/min. Gradient HPLC was used with a changing solvent ratio from 80:20 to 10:90 over 8 min and then 80:20 for 13 min for A/B, respec-tively. Quantifi cation was achieved by MS in the positive ionization mode, with ESI

as an interface, a drying gas fl ow of 10 L/min, a drying gas temperature of 350°C, a nebulizer pressure of 50 psi, a capillary voltage of 4,000 V, and a fragmentation energy of 130 V. This method obtained good precision, accuracy, and speed with an LOQ of 0.1 μg/ml and an LOD of 35 ng/ml (signal to noise ratio of 3) with a time of 8 min per analysis. It was concluded that this method could be used for the analy-sis of glucosamine in human plasma, including the analyanaly-sis of basal glucosamine plasma levels [Huang et al. 2006a]. This technique allows for information to be gathered on the physiological role of endogenous glucosamine and its involvement in disease processes.

Another method studied by Roda et al. [2006] used HPLC, followed by ESI-MS/

MS detection. In this study a polymer-based amino column was used to increase the retention of glucosamine, helping to prevent the need for a derivatization step. This is benefi cial for several reasons. First, the speed of analysis is increased. Second, derivatization reactions are aspecifi c and could derivate other plasma components, resulting in an increased “noise” in the separation and detection steps. Separation of components on the column was achieved using a gradient composed of Milli-Q water (A) and acetonitrile (B) at a fl ow rate of 0.3 ml/min. The gradient elution pro-gram used was as follows: 20% A for 7 min, 7–8 min a linear increase from 20 to 50% A, 50% A for 8 min, 16–17 min a linear decrease from 50 to 20% A, 10 min 20% A. Detection was effected using a triple quadrapole mass spectrometer that was set on the positive ionization mode, with quantifi cation performed in the multiple reaction monitoring mode. This method achieved an LOQ and LOD of 10 and 5 ng/

ml, respectively, without the need for a derivatization step. Roda et al. concluded that this method was suitable for the measurement of endogenous glucosamine plasma levels attributable to its high sensitivity. The method is said to be advantageous over other techniques because no preanalytical derivatization step is required, resulting in analysis that can be performed quickly and reduce analytical variability [Roda et al. 2006].

Both methods using HPLC coupled to ESI-MS detection give high sensitivity and accuracy. However, criticisms exist around the use of HPLC ESI-MS methods for the routine analysis of glucosamine. Some claimed that LC-MS technology is not yet widely available in laboratories, thus making the method nonsuitable for widespread analysis. It is also claimed that precolumn derivatization with FMOC-Cl with UV detection gives the same sensitivity at less cost than the LC-MS technique [Huang et al. 2006b].

The Use of HPLC with Alternative Detection Methods

HPLC with precolumn derivatization methods are cheap and effective. They offer a suitable level of accuracy for analysis of glucosamine products, and derivatization with FMOC-Cl may offer suitable sensitivity for analysis of biological samples. The derivatization step is involved with many criticisms of the method. The initial deriva-tization reaction may be time consuming, increasing the time of analysis render-ing the methods unsuitable for routine use. Once made, the derivative-glucosamine complexes could have stability issues that may confound the results. Finally, the

derivative compounds themselves could negatively affect the performance of the assay. To avoid these issues, HPLC ESI-MS/MS is available using an amino column that circumnavigates the use of derivatization agents. However, this method is more expensive and less readily available but offers good sensitivity. The last HPLC option available is the use of indirect fl uorescence detection with L-TRP or 5-MTP. This method avoids the derivatization step and is cheap and accessible; however, it lacks suitable accuracy for analysis of biological samples.

Overall, the HPLC method of choice depends on the analytical function need that is to be performed.

Gas Chromatography

Glucosamine has been determined in soil using gas chromatographic methods [Zhang 1996]. The technique is said to suitably sensitive, but the need to make solutes volatile by complicated sample preparation procedures can be time con-suming and can result in multiple peaks for a single component [Qui et al. 2006].

This makes the method unsuitable for routine use. Zhang et al. [1996] looked at the determination of glucosamine in soil samples, and, thus, its relevance to the quantifi cation of glucosamine in commercial products or plasma samples may be questionable.

High-Performance Thin-Layer Chromatography

A quantitative densitometric high-performance thin-layer chromatographic method was developed by Ester et al. [2006] for determination of glucosamine in dietary supplements. The method was sought because no rapid, simple, or selective HPLC method was reasonably available for the quantitative determination of glu-cosamine from its complex matrix. Ester et al. achieved separation on 20 × 10 cm silica gel 60 F₂₅₄ high-performance thin-layer chromatographic plates, with a mobile phase consisting of 2-propanol/ethyl acetate/ammonia solution (10:10:10 v/v/v).

Because glucosamine lacks a suitable chromophore, the plates were immersed in a visualization reagent solution consisting of diethyl-ether/glacial acetic acid/

anisaldehyde/sulphuric acid (136:91:1.2:20 v/v/v/v). The plates were then processed, and glucosamine appeared as brownish-red zones on a colorless background.

Densitometric determination of glucosamine was performed at 415 nm by refl ec-tance scanning. The amounts of glucosamine were found from the intensity of diffusely refl ected light. The method is advantageous because it circumvents the tedious and time-consuming sample preparation steps necessary for the HPLC techniques discussed above. Ester et al. [2006] concluded the method to be reli-able, repeatreli-able, and accurate.

Capillary Electrophoresis Determination

Both HPLC with UV/fl uorescence or ESI-MS detection include particular dis-advantages that are discussed above, including a relatively high cost. Capillary

electrophoresis (CE) has been considered as a suitable alternative because of its high effi ciency, fast speed, small sample requirement, simplicity, and fl exibility [Liang et al. 1999]. For such reasons, CE has been widely used for the rapid analysis of biomolecules, such as carbohydrates, amines, and amino acids, giving high resolu-tion and short separaresolu-tion times of approximately 3 min [Skelley et al. 2005]. Similar to the HPLC method with precolumn derivatization, the main drawback of CE is its low detection sensitivity to glucosamine, which has to be improved by incorporation of a suitable UV absorbing group onto the glucosamine molecule [Qi et al. 2006;

Skelley et al. 2006].

Capillary Electrophoresis with Dansylation of Glucosamine under Microwave Irradiation

Qi et al. [2006] appreciated that a major drawback of derivatization is its increase in the time of analysis. To obviate this problem, they investigated the use of dan-syl chloride as a suitable labeling agent. To accelerate the labeling process of glu-cosamine, they performed the dansylation reaction in a microwave oven, which gave labeling speeds up to 50 times faster than common methods. Derivatization was achieved by mixing 10 mg of dansyl chloride with 10 ml of acetone and the glu-cosamine solution (tablets dissolved in 80 mM borate buffer at pH 9.5). The solution was placed in a microwave oven and irradiated at 385 W for 6 min. CE was per-formed using a bare fused-silica capillary of 75 μm inner diameter × 57 cm. Samples were injected at 0.5 psi for 2 s and separated at +18 kV at 20°C. The separated bands were detected by UV absorption at 214 nm. This technique gave reasonable sensitiv-ity with an LOD of 1 μg/ml. Qi et al. concluded that the use of CE using accelerated labeling of glucosamine with dansyl chloride under microwave irradiation was a reliable, accurate, quantitative, and highly applicable method for determination of glucosamine content within commercially available glucosamine tablets but is not suffi ciently sensitive for determination of glucosamine in biological samples [Esters et al. 2006].

Microchip Capillary Electrophoresis with Fluorescamine Labeling for Anomeric Composition Determination

Glucosamine can exist as either an alpha or beta anomer, an anomer being an epimer that is a stereoisomer of a saccharide differing only at the reducing carbon atom. Because of this, there is a need to assess the anomeric composition of glucosamine products and to assess the interconversion rates between the two anomers. Skelleyand Mathies [2006] developed a microchip capillary electrophoretic method able to resolve both the alpha and beta anomers of glucosamine, allowing for determination of the anomeric composition of a sample. Previous chromatographic methods reported timescales of analysis between 10 and 20 min, which led to poor resolution between the alpha and beta anomers; however, because of the inherent speed of CE, this did not occur. The blurring in resolution occurs as a result of on-column mutarotation between the two anomers of glucosamine; thus, the faster

the analytical technique the less time mutarotation has to occur and less blurring of resolution is seen. Skelley et al. labeled glucosamine with fl uorescamine by mixing 2 μl of sample with 20 mM fl uorescamine in dimethylsulfoxide. The samples were analyzed at room temperature using all-glass microfabricated devices made in-house.

The CE separations were performed at 700 V/cm on a portable CE instrument with a 100-μm-diameter fi ber optic-coupled photomultiplier tube for fl uorescence detection.

The inherent speed of microchip CE enabled Skelley et al. to observe the alpha and beta anomers of glucosamine and their interconversion rates in real time. It was noted that the interconversion between the anomers may have been effected by the fl uorescamine labels. Furthermore, if interconversion was allowed to take place before labeling, more accurate results could be acquired. However, this was at the expense of time, and, if labeling took place after mutarotation, the time of overall analysis rose from approximately 2 min up to 2 h. Skelley and Mathies [2006] concluded that this method was fast, portable, and suitable for glucosamine determination.

The Pros and Cons of Capillary Electrophoresis

The Pros and Cons of Capillary Electrophoresis