Development of a thin layer chromatography-based method to detect sorbitol presence in milk and its applicability in formalin preserved milk samples

Abstract

Sorbitol has been the new and emerging adulterant in dairy industry. The main aim of the study was to develop a method to detect sorbitol in milk, which is not affected by other sugars, polyols and formalin. Hence, a thin layer chromatographic (TLC) method was standardized to detect the sorbitol in milk. In the study 90 s duration for the impregnation of Silica gel 60F TLC plates with Cu- ions was found suitable to resolve sorbitol as a distinct spot. The standardized conditions were (1) developing solvent system consisting of n-propanol: ethyl acetate: water (7:1:2), (2) 0.5% of potassium permanganate in 0.1 M NaOH as color developing reagent. (3) Drying temperature (65°C/ 10 min.) after spraying the color developing reagent. The limit of detection was 0.2% of added sorbitol in milk. The standardized method could also detect the sorbitol in the presence of sucrose, glucose and polyols like mannitol and maltitol. In both cow and buffalo milk samples the standardized methodology performed well in detection of sorbitol. The method also performed well in sorbitol spiked formalin preserved milk samples. This method can be an alternative to the other methods involving costly equipment in detecting adulteration of milk with sorbitol.

Introduction

Milk has long been known as a source of various nutrients essential for a proper growth in human beings. It is considered as a complete food due to its balanced nutritional value in terms of energy, protein, carbohydrate, fat, vitamins etc. (Park 2009). It is known fact that apart from milk being consumed in liquid form, it is also used as raw material for various dairy products. In order to fulfil the ever-growing demand of milk, the suppliers have found it profitable to adulterate milk with cheaper chemicals having harmful health effects, the incidents of the malpractice increases during summers and peak festive seasons. Consumption of adulterated milk does not fulfil nutritional benefits of pure milk and can cause long and short-term health problems such as diarrhoea, headaches, eyesight problems, hypertension, kidney stones, and even death (Luther et al. 2017; Handford et al. 2016). Water was reported the most common adulterant even in the executive summary of National Survey on Milk Adulteration, India (Anon 2012). The most common practice till date is the dilution of milk with water and then adjustment of its solids- not-fat (SNF) content/lactometer reading by addition of cheaper chemicals like urea, cane sugar, starch, sulfates and common salts (Azad and Ahmad 2016). National Milk Safety and Quality Survey revealed that there was non-compliance on account of low fat or low SNF, two key quality parameters both in raw and processed milk. The probable reason reported was either due to the poor farm management practices or due to dilution of milk with water (Anon 2019). Compositional and adulteration analysis carried out in ten different towns of Lahore, Pakistan reported that none of the collected milk samples were positive for sorbitol (Shehzadi et al 2016). Similarly, in another study it was reported that out of ten samples of milk collected from local market of Lahore, six were found to have sorbitol (Basharat et al., 2019). The recent experience of the industry tells that unscrupulous traders have also modified their strategy to adjust the SNF content of diluted milk. The probable reason could be the emergence of rapid test kits to detect the common milk adulterants like urea, carbohydrates, sucrose, glucose, starch by institutions like National Dairy Research Institute, Karnal (Haryana) and National Dairy Development Board, Anand (Gujarat). The most talked about recent adulterant in dairy circle is the sorbitol. Industry and academia both are struggling to check the adulteration of milk with this emerging adulterant. Recently, a patent to detect sorbitol in milk has been granted (Mother Dairy Fruits and Vegetable Pvt. Limited, 2016). The patented method involves boiling/coagulation/filtration/incubation steps and its details are not available in the public domain. Another method used to detect sorbitol (Chaudhary et al. 2015) in the survey study carried out in Pakistan is based on sorbitol’s reaction with ferric sulphate and sodium hydroxide, but details of the method have not been reported by the authors. The detection limit of the said method was 0.25%. Similarly, another rapid and simple dye-based method (Karra 2021), was reported to detect sorbitol addition in milk, wherein the reported limit of detection was 0.5%. However, in all the cases the interference of other polyols (mannitol and maltitol), maltodextrin, sugars and adulterant has not been reported. It is well established that adulterators use the concoctions of different adulterants to mask the tests. As per Food Safety and Standards (Laboratory and sample analysis) regulation, formalin has been recommended to preserve milk of any type, wherein formalin in the proportion of 0.1 ml for 25 ml or 25 g is allowed (FSSAI 2011). This is allowed for the samples to be preserved before their dispatch to a Notified/ referral laboratory for testing. Therefore, in the present investigation an attempt was made to (1) develop a sensitive, specific and cost effective chromatographic method i.e. TLC method based on the property of polyols and sugars to form complex with metal ions (Hadzija et al. 1994). (2) Detection of sorbitol adulteration in milk even in presence of other polyols and sugars. The efficacy of the developed method was also tested in formalin preserved milk samples.

Materials and methods

Chemicals and reagents

Acetone, ammonia-25%, ethyl acetate, n-propanol, sucrose were purchased from Merck Specialities Pvt. Ltd, Mumbai-India; boric acid and urea were procured from Thomas Baker Chemicals Ltd, Mumbai. D-sorbitol was purchased from Sigma Aldrich, India; lactose monohydrate and mannitol were purchased from Himedia laboratories Pvt. Ltd Nashik; maltitol was procured from Tokyo chemicals industry, Japan. Cupric acetate- Cu (CH₃OO)₂. H₂O was purchased from Central Drug House Pvt. Ltd, New Delhi. Ethanol absolute was purchased from Jiangsu Huxari International Trade Co. Ltd., China; Formalin and Silica gel 60F TLC plates were purchased from SD Fine Chemical limited, Mumbai. Glucose was procured from Qualigen Fine Chemicals, Mumbai, India, Sodium hydroxide and Potassium dihydrogen orthophosphate was procured from Thermofisher scientific India Pvt. Ltd, Mumbai and Whatman filter paper (NO. 42) was from GE healthcare, UK Ltd.

Alkaline potassium permanganate solution 0.5%w/v in 0.1N NaOH.

Aqueous solution (0.2%) of sorbitol, lactose, glucose, mannitol, maltitol and their mixture were used to standardize and optimize the solvent system and cu- ion impregnation time.

To check the detection level of sorbitol, aqueous solution of sorbitol having concentration @ 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 was prepared.

Preparation of sorbitol spiked milk samples

Fresh pooled cow milk samples having fat percentage 3.8 ± 0.12 and solids -not- fat percentage 9.0 ± 0.32 and pooled buffalo milk samples having fat percentage 7.5 ± 0.37 and solids -not- fat percentage 9.9 ± 0.16 were collected from Livestock Research Centre, National Dairy Research Institute, Karnal. To standardize the TLC- based methodology of sorbitol detection, cow milk samples were spiked @ 0.5 and 1.0% with sorbitol and sorbitol in conjunction with glucose, sucrose, maltitol, mannitol.

To check the effect of formalin on the standardized methodology, both cow and buffalo milk samples were spiked with (i) sorbitol alone @ 0.5 and 1% (ii) sorbitol in conjunction with mannitol and maltitol having 0.5% concentration of each. These samples were then preserved by adding formalin @0.4% as a sample preservative and stored at 4℃ and 30℃. The samples were then tested for sorbitol presence by using the developed TLC method on zero day i.e. day of the start of the experiment and on 30^th day.

Preparation of copper impregnated (Cu-impregnated) TLC plates

The Cupper impregnated silica gel-60 plates were prepared essentially as per the method described by (Hadžija et al. 1994), with a slight modification. The modification done was the impregnation time, wherein impregnation with copper was done for 90 s instead of 30 s. Then plates were taken out, shifted to the oven maintained at 100°C and allowed to dry. The prepared plates were stored in a cardboard cover and kept in dark till their use. The modification was necessitated as 30 s impregnation of TLC plate did not result into the appropriate separation of sorbitol from other sugars and polyols.

Detection of sorbitol in aqueous system using Cu-impregnated TLC plates

2 µl of each prepared sample was applied on activated Cu- impregnated TLC plate. Then, the samples on plates were dried by using hot air blower. The dried plates were kept in a TLC chamber which was already saturated with standardised i.e. n-propanol: ethyl acetate: water (7:1:2). After completion of the run i.e. about 2 h, the plates were dried at 70°C for 15 min in a hot air oven. The dried plates were dipped in freshly prepared 0.5% (w/v) alkaline potassium permanganate solution for exactly 30 s. Again, plates were dried at 65°C for 10 min in an oven, the appearance of yellowish spots agaist violet background showed the movement of different sugars and sorbitol.

Detection of sorbitol in milk system using Cu- impregnated TLC plates

The standardised conditions for the detection of sorbitol in aqueous system were evaluated in milk system, where in milk was spiked with sorbitol @ 0.1, 0.2, 0.5 and 1.0%. To avoid the interference of casein in the analysis, the milk was made free of casein by coagulation with acetone. 2.0 ml of sugars spiked milk and 4 ml of acetone were taken in 15 ml centrifuge tubes, vortexed for 5 min and centrifuged at 1500 RPM/5 min (Karra 2021). Supernatant obtained was filtered through Whatman no.42 filter paper. Filtrate was diluted with water in three dilutions, 1:2, 1:3 and 1:4. 2.0 µl of the each dilution was applied on Cu-impregnated TLC plate, and plates were developed as per the procedure detailed out for aqueous solutions.

Results and discussion

Effect of developing solvent composition on sorbitol separation in cu- impregnated TLC

Polyols and sugars form complex with metal ions (Hadzija et al. 1994). This property was explored by Hadžija et al. (1994) for the separation of sorbitol and glucose in extracts of human erythrocytes, wherein copper- impregnated silica gel F60 TLC plates, developing solvent i.e. n-propanol: water (4:1) and detecting reagent i.e. alkaline potassium permanganate was used. In the present investigation, attempt was made to standardize a TLC method based on the principle of the above said method to separate newly emerged milk adulterant i.e. sorbitol from other polyols and sugars having the likelihood of adulteration in milk. To achieve this aim, developing solvent having varied composition (Fig. 3) and duration of cu- ions impregnation on TLC plates were optimized to separate sorbitol from other polyols.

Fig. 3.

a Effect of copper ions impregnation time of silica gel F60 plates on separation of sorbitol , sugars and polyols using developing solvent system. n-propanol, ethyl acetate, water (7:1:2). b Effect of sorbitol concentration on the appearance of yellow spot/ streak on cu-impregnated silica gel F60 plates ( lane1-6 representing concentration of 0.1%, 0.2%,0.5%, 1%,2% and 0.05%

It is evident in the TLC chromatogram (Fig. 1) that sorbitol showed difference in the movement than other studied sugars and polyols on silica gel F60 TLC plate impregnated with cu- ions for 30 s and developed in a mixture (4:1) of n-propanol: water (Hadžija et al. 1994). The movement of sorbitol was slow in comparison to glucose and lactose but there was a considerable streaking. Movement of mannitol and maltitol was also studied. It is clear from the chromatogram (i) in Fig. 1 that both mannitol and maltitol travelled a higher distance from the origin than the sorbitol. Maltitol moved slightly ahead of sorbitol. Mannitol moved ahead of sorbitol and maltitol, but close to the distance to which lactose moved. At the same time the streaking was more in case of mannitol. In case of mixture (Mx) some separation could be achieved but it was not satisfactory due to streaking. Since, the resolution was not upto the mark and there was prominent streaking, hence Rf-values were not measured and simply by observing the movement pattern the study was further extended to find out a suitable combination of solvents. During the study, developing solvents with varying composition and concentration i.e. n-propanol: ethyl acetate: distilled water (7:1:2, 4:1:2 and 5:1:2), n-propanol: ethyl acetate: ammonia: water (4:2:1:1), n-propanol: acetonitrile: water (4:2:1) and n-Butanol: acetic acid: ethyl ether: water (9:6:3:1) were tried to attain the better resolution of polyols. It is evident from chromatogram ii (Fig. 1), that movement of sorbitol in this developing solvent just reversed and it moved towards the top of the TLC plate. However, in the mixture of glucose + lactose + sorbitol, the distance travelled by sorbitol was very close to the distance travelled by mannitol and maltitol. In this case, also the resolution in the mixture was very poor and distinction between the compound of interest i.e. sorbitol from mannitol and maltitol was not possible. In other tried systems barring n-propanol: ethyl acetate: water (4:2:1) the resolution on cu- impregnated TLC plate was not so efficient. It is also evident from the chromatograms iii & iv (Fig. 1), that introduction of other solvent in the developing solvent did not yield any improvement. However, the introduction of ethyl acetate in the developing solvent i.e. n-propanol: ethyl acetate: water (4:2:1), the resolution of sorbitol was relatively better as indicated in TLC chromatogram number five (Fig. 1). Therefore, the above mentioned developing solvent was considered as a baseline and further optimization of the proportion of constituent solvents was carried out.

Fig. 1.

Effect of solvent system on the separation of aqueous solutions (0.2%) of polyols and sugars. (i) n-propanol: water (4:1) (ii) n-propanol, ethyl acetate, ammonia and water (4:2:1:1), (iii) n-Butanol, acetic acid, ethyl ether, water (9:6:3:1), (iv) n-propanol, acetonitrile water (4:2:1), (v) n-propanol, ethyl acetate, water (4:2:1)

Optimization of the proportion of constituent solvents to improve sorbitol separation on cu- impregnated TLC

It is evident from the chromatograms i & iii (Fig. 2) that the developing solvent consisting of n-propanol: ethyl acetate: water in proportion 5:1:2 & 7:1:2 performed equally well in resolving the sorbitol from other sugars and polyols. The movement of sorbitol in both the developing solvent systems was found to be slower than other sugars and polyols. This could be due to the fact that glucose and disaccharides formed weak complexes with copper ions and hence moved towards the top of the TLC plate. It is also evident from the TLC chromatogram that mannitol and maltitol also formed complexes with impregnated copper ions on TLC plate but their complexes were not as strong as that of sorbitol hence travelled upwards and formed a streak. However, the movement of polyols was not as fast as that of mono and disaccharides i.e. glucose, lactose and sucrose. Looking at the movement patterns of different sugars and polyols, it is evident that the movement of the sorbitol was less. In mixture also the sorbitol remained confined near the sample application point and resolved well on cu- impregnated TLC plate.

Fig. 2.

Optimization of the proportion of n-propanol, ethyl acetate and water in developing solvent system. (i) n-propanol, ethyl acetate, water (5:1:2) (ii) n-propanol, ethyl acetate, water (5:1:3) (iii) n-propanol, ethyl acetate, water (7:1:2)

Effect of duration of copper impregnation on sorbitol separation in TLC

From the results presented in above discussion, it is evident that polyols have the ability to form relatively strong complexes with copper than sugars like glucose, lactose and sucrose. Moreover, literature also suggested that for better complex formation 3 mol of copper and 1 mol of sorbitol was the better ratio (Imamura et al. 1978). Therefore, in the present study attempt was also made to optimize the copper ions impregnation by varying the dipping time of Silica gel 60F TLC plates in copper impregnation solution. The developing solvent system used was n-propanol: ethyl acetate: water, (7:1:2). The chromatogram (Fig. 3A) showed that in case of TLC plate impregnated with cu- ions for 30 s, the streaking of sorbitol was more followed by mannitol and maltitol. Since the streaking was very prominent in sorbitol, hence Rf value measurement was of no use. This phenomenon could be attributed to the possibility of lower concentration of copper ions impregnated on the TLC plate. This could be the reason that less time of impregnation might have led to lower concentration of copper ions in silica gel plate surface. The Rf values of glucose, lactose, mannitol and maltitol were 0.80, 0.57, 0.70 and 0.55, respectively. As evident from the TLC chromatogram-iii (Fig. 3A), wherein plates were impregnated for 15 min, all the studied polyols and sugars remained at the bottom of the plate except for little movement in the case of glucose and lactose. This could be due to the high concentration of copper ions impregnated on TLC plate as a result of longer duration of dipping in the cu- impregnation solution, which might have led to strong complex formation between the polyols, irrespective of the type of polyols. However, in case of TLC plate which was impregnated for 90 s and represented as TLC chromatogram -ii (Fig. 3A), the sugars (glucose, lactose and sucrose) got separated clearly from each other as well as sorbitol. The Rf values of glucose, lactose and sucrose were 0.78, 0.55, and 0.72, respectively. Sorbitol showed Rf value of 0.23, which is also evident from the fact that sorbitol remained as dark yellow small streak against violet background close to the point of sample application. On the contrary, mannitol and maltitol had Rf value of 0.42 and 0.34 which is apparent from the chromatogram also as both mannitol and maltitol moved ahead of sorbitol. It is also evident that both maltitol and mannitol showed some streaking but their maximum content moved faster and far ahead of sorbitol. It is also evident from the results that though manitol’s movement was a bit faster than maltitol but their chances of mixing with each other in the mixture of these two looked more. However, they were well moved ahead of sorbitol, even in the mixture of sorbitol, glucose, lactose, sucrose, maltitol and mannitol. It is clear from the chromatogram -ii ( Fig. 3A) that sorbitol remained close to the point of the application of sample as evident from lane seven in chromatogram -ii ( Fig. 3A). This showed that 90 s impregnation resulted into the optimum concentration of copper ions in silica gel, which was sufficient to prevent excessive streaking and optimal separation of polyols (clear separation between sorbitol, mannitol and maltitol) and low enough to prevent the formation of undesirable stronger complexes between sugars and copper ions. Hence, the impregnation time of 90 s was finally selected and adopted in the subsequent experiments to detect sorbitol in milk system. The TLC based method was found to have an advantage of detection sorbitol even in the presence of other polyols, unlike color based method of sorbitol detection in milk (Karra 2021), wherein the minimum level of added sorbitol detection was 0.5%. However, the interference of mannitol was not been reported. Similarly (Aslam et al. 2019) reported minimum level of added sorbitol detection @ 0.25%, but effect of other sugars and polyols was not reported in the study.

Effect of sorbitol concentration on the behaviour of its movement on copper impregnated TLC plate

It is evident from the TLC chromatogram (Fig. 3B) lanes 1, 2 and 6 that a clear yellow spot against violet background was visible with minimum streaking. It is also evident from the results a faint spot of yellow color was observed near the spot of the sample application in case of aquesous solution of sorbitol having a concentration of 0.05%. It is also evident from the results of TLC lane 3,4 and 5 of the chromatogram (Fig. 3B) that as the concentration of sorbitol in the aqueous solution was increased the intensity of yellow color increased and at very high concentration i.e. more than 0.2% the extent of streaking on the plate also increased (Fig. 3B). The results of cu- impregnated TLC clearly showed that the developed TLC method has the potential to detect sorbitol in milk.

Evaluation of the developed TLC method in sorbitol spiked cow milk samples

It is the basic fact that for any chromatographic analysis, the interfering compounds need to be removed from the sample matrix. However, in this case it was difficult to remove lactose being in true solution phase, therefore the proteins were removed and deproteinated filtrate containing native lactose, salts and added sorbitol was used. It was observed that on applying the deproteinated filtrate of sorbitol spiked milk sample on Cu-impregnated TLC plates followed by development in standardized TLC conditions, lactose appeared as a broad thick band on TLC plate as depicted in lane 1&2 of the chromatogram (Fig. 4). This can be attributed to the very high concentration of lactose in the filtrate, which might have led to a streak formation instead of clean separated spot on the TLC plate. To take care of this interference of the lactose, effect of filtrate dilution was studied, wherein cow milk filtrate obtained from the blank was diluted with distilled water in 1:1, 1:2, 1:3 and 1:4 ratio. It is evident from the chromatogram (Fig. 4) lane 3, 4 &5 that as the dilution level was increased the streaking of lactose was decreased considerably. It is also evident from the results depicted in TLC chromatogram (Fig. 4) lane 8 that in case of filtrate obtained from the 0.1% sorbitol spiked cow milk followed by dilution in 1:2 with water, a small spot of sorbitol was visible near the point of application of the sample. In case of the filtrate obtained from 0.2% sorbitol spiked milk and diluted to 1:2 ratio (lane 11; Fig. 4), the spot of sorbitol was very evident and clear. Similarly, in case of samples spiked with higher level of sorbitol i.e. 0.5% and 1% the visibility of the sorbitol spots was very good even at higher dilutions as evident in lane 17 & 21 of the TLC chromatogram (Fig. 4). This led to the conclusion that in case of unknown samples of milk it is advisable to dilute the filtrate at least in 1:2, 1:3 dilution. This will take care of the sorbitol detection in milk even at higher levels.

Fig. 4.

Effect of dilution of sorbitol spiked cow milk filtrate on the clarity of separation of sorbitol on cu- impregnated silica gel F60 plates; (1) blank without dilution (2) blank (1:1) dilution (3) blank (1:2) dilution (4) blank (1:3) dilution (5) blank (1:4) dilution, (6) 0.1% sorbitol spiked no dilution (7) 0.1% sorbitol spiked (1:1) dilution (8) 0.1% sorbitol spiked (1:2) dilution (9) 0.2% sorbitol spiked no dilution (10) 0.2% sorbitol spiked (1:1) dilution (11) 0.2% sorbitol spiked (1:2) dilution (12) 0.5% sorbitol spiked no dilution (13) sorbitol* standard (0.2%) (14) 0.5% sorbitol spiked no dilution (15) 0.5% sorbitol spiked (1:1) dilution (16) 0.5% sorbitol spiked (1:2) dilution (17) 0.5% sorbitol spiked (1:3) dilution (18) 1% sorbitol spiked no dilution (19) 1% sorbitol spiked (1:1) dilution (20) 1% sorbitol spiked (1:2) dilution (21) 1% sorbitol spiked (1:3) dilution (22) 1% sorbitol spiked (1:4) dilution (23) sorbitol standard (0.2%)

Effect of formalin (0.4%) as milk sample preservative on the developed TLC- method of sorbitol detection

Formalin (0.4%) is generally used as a preservative to store the milk samples lifted up by the food safety officers (FSO) from the open market/ processing facility before their analysis. Therefore, the effect of the formalin (0.4%) on the developed method was also studied in both cow and buffalo milk preserved with formalin and stored at room temperature (30 ± 1℃) and at refrigerated storage i.e. 4℃. Refrigerated samples were analysed on the beginning of the experiment (zero day) and 30^th day of storage. It is evident from the results depicted in lanes 2, 3, 4, 6, 7 & 8 of chromatograms (Fig. 5A) that a clear spot of sorbitol appeared at the site of application of the sample unlike blank where in there was no spot at the site of the sample application ( lane 1& 5 of Fig. 5A). Even in case of mixture of polyols, the sorbitol spot was clearly visible as evident in lane 4&8 of the chromatogram (Fig. 5A). As evident from the results depicted in the chromatograms (Fig. 5B), that in case of milk samples spiked with sorbitol and subsequently preserved with formalin, the sorbitol spot appeared near the site of the sample application even after thirty days of storage at both 4℃ as well as 30℃. These finding clearly showed that formalin used as sample preservative did not show any adverse effect on separation behaviour of sorbitol and the developed TLC method found effective in detecting the sorbitol. Hence, it can be concluded that the developed cu- impregnated TLC method has the potential of its adoption in State Food Laboratories/ Referral Food Laboratories.

Fig. 5.

a Effect of formalin preservation on zero day storage of sorbitol spiked cow and buffalo milk samples on separation of sorbitol on cu- impregnated silica gel F60 { (1) Cow milk (2) 0.5% sorbitol spiked cow milk (3) 1% sorbitol spiked cow milk (4) sorbitol+mannitol+maltitol spiked (0.5% each) cow milk. (5) Buffalo milk (6) 0.5% sorbitol spiked Buffalo milk (7) 1% sorbitol spiked Buffalo milk (8) sorbitol+mannitol+maltitol spiked (0.5% each) Buffalo milk }. b Effect of formalin preservation on 30th day storage of sorbitol spiked cow and buffalo milk samples on separation of sorbitol on cu- impregnated silica gel F60 { (1) Cow milk (2) 0.5% sorbitol spiked cow milk (3) 1% sorbitol spiked cow milk (4) sorbitol+mannitol+maltitol spiked (0.5% each) cow milk, (5) Buffalo milk (6) 0.5% sorbitol spiked Buffalo milk (7) 1% sorbitol spiked Buffalo milk (8) sorbitol+mannitol+maltitol spiked (0.5% each) Buffalo milk}

Effect of other sugars and sugar alcohols on the separation of sorbitol in milk filtrate obtained from the sorbitol spiked cow milk sample

To mask the test of sugar detection the unscrupulous persons involved in the dairy sector follow the practice of using the concoctions of adulterants like sugars/polyols. Keeping the said fact in view, the standardised TLC method was also evaluated to detect the sorbitol in the presence of other sugars, such as glucose, sucrose, mannitol and maltitol. As evident from the results depicted in chromatogram (Fig. 6) of milk filtrate, sorbitol spot was clearly visible near the point of application of the sample. To make the study more effective, concentrations of sugars at the level of 0.5% and 1%, along with same concentration of sorbitol was spiked in milk and detected through the standardised TLC method. It is evident from the study that sugars like glucose and sucrose did not show any interference in the developed method. Similarly, mannitol and maltitol also moved away from the point of the sample application and did not interfere with the movement of sorbitol. The sorbitol spot appeared in the close vicinity of the point of sample application on the TLC plate. As expected the mannitol and maltitol merged together which can be seen in lanes 10 & 11 of the chromatogram (Fig. 6). Therefore, it can be concluded that sorbitol added to milk could easily be detected by the standardised TLC method even in the presence of other sugars and polyols.

Fig. 6.

TLC chromatogram of sugars and polyols combinations (0.5% and 1% each) separated using n-propanol, ethyl acetate, water (7:1:2) as solvent on Cu-impregnated plates, milk (1) blank, (2) & (3) G+S, (4) & (5) Su +S, (6) & (7) MN + S, (8) & (9) ML +S, (10) & (11) G + L + S + MN + ML

Conclusion

TLC-based method has been developed to detect sorbitol presence in milk. The method has the capability to detect sorbitol to the level of 0.2% and above in milk and is not affected by the presence of studied levels of lactose, glucose, sucrose, maltitol and mannitol i.e. 0.5% & 1.0% maximum. The efficiency of developed method remained unaffected in formalin (@ 0.4%) preserved milk samples. Therefore, it was concluded from the study that the developed method had the potential to be used as a test method to detect sorbitol in the presence other polyols, sugars and formalin. The developed method is cost effective and does not involve any high end costly instrumentation like high performance liquid chromatography (HPLC).

Author contributions

RK: Experimental bench work was carried out. PM: Preliminary work related to TLC and extraction protocol. PSR: Reviewing and standardization of extraction protocol. RS: Compilation of the data for graphical presentations. SA: Reviewing and editing. VS: Conceptualization of the problem, compilation of the manuscripts and logistic support.

Funding

The project was a student project, which was supported by the Institute.

Data availability

The authors declare that the data supporting the findings of this study are available within the article.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors have neither any conflicts of interest nor competing interest to declare.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

The work is a bonafied work carried out under a master’s program and the authors give their consent to publish the work and images included in the manuscript.