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Full Research Article

Quantification of Methylglyoxal in Honey using LC-MS Method

K
K. Sonia1,*
M
M.A. Suman Raj1
P
P. Dharshini Priya1
S
S. Sabarish1
R
R. Kaviya1
S
S. Sayalee1
S
S. Sayalee1
1Department of Pharmaceutical Chemistry, Sri Ramachandra Faculty of Pharmacy, SRIHER(DU), Porur, Chennai Chennai-600 116, Tamil Nadu, India.

ABSTRACT

Background: Honey is sweet and viscous substance produced mainly by honey bees. It contains simple sugars like glucose and fructose, making it sweet and easily digestible.

Methods: The present study aimed to provides a comprehensive and critical review of the different types of adulteration, common sugar adulterants and detection methods and draws a clear perspective toward the impact of honey adulteration on human health.

Result: The MGO can be identified and quantified in different honey samples within the concentration range of 50 mg to 600 mg/kg. This study investigated the release pattern of MGO from five honey-based (Lepto- spermum spp.) commercial products and the honey matrix itself. The conversion of MGO into the more stable compound 2-methylquinoxaline allows for an easy evaluation of the MGO content. The recovery study showed a detectable MGO amount of around 90%. The correlated MGO amount in different samples of honey was calculated accordingly. Overall, there is no release pattern for MGO was noticed from the honey matrix as well as from all investigated commercial products.

INTRODUCTION

Honey is identified as a pleasant and fresh edibles so, feasible absorbed as a sweetener in addition to as medicine due to its curative effect on human health (Atrott et al., 2012). It is vulnerable  to adulterants give rise to by humans that operate the quality of honey (Saranraj et al., 2016). Despite the fact that honey consumption has significantly expanded globally in new years, its safety is not routinely evaluated and tracked (Irish et al., 2011). Their expectation and significance in this high quality  product has decreased. Since the sum total of consumers of honey have enlarged in new years, (Itoh et al., 2019). Honey debasement are any material that are added to the pure honey in Fig 1 (Fakhlaei et al., 2020) In this sense, this study offers a thorough and critical analysis of the many forms of defilement, typical sugar adulterants and detection techniques. It also presents a clear account of the consequence of honey adulteration on human health (Kwok et al., 2016). Adulteration causes the consumer’s blood sugar levels to rise, whatever can lead to hypertension, diabetes, obesity and abdominal increase in body mass (Majtan et al., 2012 and Pappalardo et al., 2016).

Fig 1: Different brands of honey sample.


       
According to multiple in vivo research designs, the liver is most commonly affected by honey adulterants, followed by the kidney, heart and brain (Saikaly et al., 2017).
       
Methylglyoxal (MG) (Fig 2) is created by the disintegrating of glycolytic intermediates, by lipid peroxidation systems, in the oxidation of acetone and the catabolism of amino acids. As a highly reactive α-oxoaldehyde, it has strong interactions with proteins and nucleic acids., causing mutations and apoptotic cell death, or generating Advanced glycation end products (AGE) that take part in the patho-physiology of issues related to diabetes as well as the develop-ment of a number of age-related illnesses, cardio vascular disease, malignant and Alzheimer’s disease, cancer (Stephens et al., 2010). The aim of the analysis is to quantify methylglyoxal present  in different brands of honey available in India, using LC-MS Method and plan of work shown in Fig 3.

Fig 2: Structure of methyl glyoxal.



Fig 3: Plan of work.

MATERIALS AND METHODS

Materials
 
The different brands of Honey were procured from the   local supermarket.  The Methyl glyoxal were purchased from MP Biologicals. The instrument used is Digital Balance - Sartorius group.
 
Preparation of buffer
 
Add 200 ml of water, to 500 ml of volumetric flask, 1 ml of formic acid and made volume up with HPLC grade water.

Mobile phase preparation
 
Before use of mobile phase, 20 ml of buffer, 80 ml of HPLC-grade acetonitrile and methanol in a 20:80 v/v ratio were combined and shaken.

Preparation of Standard
 
Weighed amounts of 2 mg for each MGO was dissolved in methanol; further dilutions are made in Methanol: Water (50:50% V/V).
 
Standard stock preparation in diluents
 
Methanol: water (HPLC grade) (50: 50% v/v).

Sample extraction procedure
 
500 µL of the Honey samples were used for sample preparation procedure. To this sample 500 µL of Phenylene diamine reagent was added. Mixture was vortexed well for few seconds and sample kept in surrounding temperature for 60 mins for completion of reaction. From above reaction mixture 50 µL of above mixture was taken and mixed to 1.5 mL of methanol and then vortexed well. Transferred the sample in to injector vial for UFLCMS analysis. The study  was bring out using UFLC-MS/MS.
 
Chromatographic conditions
 
The temperature of the auto sampler retain  at 40oC.Before being utilized, the mobile phase remained ultrasonically degassed subsequently going all the time a 0.22 m Millipore membrane filter. LC operated for six minutes while the column oven 40oC was retained. HPLC-grade water and acetonitrile made up the rinse solution. (80:20, v/v) (Sravani et al., 2024).
       
API 4000 UFLC-MS/MS equipment were pre-ownfor the analysis.(Linear triple quadrupole Mass Spectrometer), which was equipped with an auto-injector. The Analyst 1.6.3 program managed the mass data collection and processing and the specifics are given as follows in (Table 1). The operating conditions for the mass spectrometer in positive mode were as mentioned in (Table 2).

Table 1: LC-MS conditions triple quadrupole LC/MS/MS mass spectrometer.



Table 2: LC-MS chromatographic conditions for methyl glyoxal.



Development and optimization of the LC method
 
The mobile phase was altered to provide adequate selectivity and sensitivity in order to achieve the desired chromatographic conditions. in a short dissociation time. Fig 4 to 11 display the results of the standard solution (Sandhu et al., 2025 and Jayasakthi et al., 2025). Methanol was used to increase peak symmetry and save analysis time. Columns from various sources were assessed and the Inertsil ODS C18, 150 mm, 4.6 mm and 5 µm analytical column was selected, because it produced appropriate peak properties, such as tailing factor and the best chromatographic performance. Moreover, acceptable resolution of MGO was applied, verifying the proposed method’s capacity to demonstrate stability. An appropriate peak symmetry and a steady baseline and a reasonable separation were attained with mL/min. It is tabulated in Table 3 and Fig 12 to 16.

Table 3: Quantitate Mode of the standard and Sample



Fig 4: Chromatogram of standard A.



Fig 5: Chromatogram of Standard B.



Fig 6: Chromatogram of standard C.



Fig 7: Chromatogram of standard D.



Fig 8: Chromatogram of standard E.



Fig 9: Chromatogram of Standard F.



Fig 10: Chromatogram of standard G.



Fig 11: Chromatogram of standard H.



Fig 12: Chromatogram of annai honey sample.



Fig 13: Chromatogram of apis himalaya honey sample.



Fig 14: Chromatogram of kodaikannal honey sample.



Fig 15: Chromatogram of lion kashmir honey sample.



Fig 16: Chromatogram of saffola honey sample.

RESULTS AND DISCUSSION

The Analyst 1.6.3 program managed the mass data’s acquisition and processing; the specifics are listed in Table 1. The mass spectrometer was operating in positive mode under the settings listed in Table 2. The proposed method’s capacity to demonstrate stability was confirmed by the achievement of an appropriate resolution of MGO. Using mL/min, a good separation with a stable baseline and good peak symmetry was obtained and tabulated in Table 3. (Sravani et al., 2024).

CONCLUSION

The MGO in different honey samples was found to be within the range of 50 mg to 600 mg/kg concentration. This study examined the MGO release pattern from five commercial honey-based (Lepto-spermum spp.) products as well as the honey matrix. The MGO in the sample can be easy to evaluate because it can be changed into the more stable compound 2-methylquinoxaline. The recovery study found that the MGO level was about 90% detectable. The linked MGO content in various honey samples was computed effectively. Despite the supplier’s failure to establish any evidence of MGO concentration. Overall, neither the honey matrix nor any of the commercial goods under investigation showed any MGO release patterns. Although it does not seem to have an effect on the total amount of MGO released, it was shown that the honey matrix influences the rate of release in addition to the potential impact of formulation excipients on MGO.
       
The analysis of MGO was difficult and complicated food matrix, such as honey, which was explained. In conclusion, methylglyoxal can be rapidly and simply quantified using UFLC-MS. This application highlights the key advantages of the method, including rapid sample preparation, strong tolerance to complex matrices and high throughput.

ACKNOWLEDGEMENT

The present study was supported by Sri Ramachandra Faculty of Pharmacy, SRIHER(DU).

Disclaimers
 
The opinions in this article belong entirely to the authors and do not reflect the official position of their affiliated organizations. While the authors aim for accuracy, they are not liable for any losses or damages caused by using this information.
 
Informed consent
 
There is no animal used for experiments.

CONFLICT OF INTEREST

The authors declare no financial or non-financial conflicts of interest. This research was conducted independently without any external sponsorship influencing its findings or publication.

REFERENCES


  1. Atrott, J., Haberlau, S. and Henle, T. (2012). Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey. Carbohydrate Research. 361: 7-11. https://doi.org/10.1016/j.carres.2012.07.025.

  2. Burns, D.T., Dillon, A., Warren, J. and Walker, M.J. (2018). A Critical review of the factors available for the identification and determination of mβnuka honey. Food Analytical Methods. 11: 1561-1567. https://doi.org/10.1007/s12161- 018-1154-9.

  3. Fakhlaei, R., Selamat, J., Khatib, A., Razis, A.F.A., Sukor, R., Ahmad, S. and Babadi, A.A. (2020). The toxic impact of honey adulteration: A review. Foods. 9(11): 1538. doi: 10. 3390/ foods9111538. PMID: 33114468; PMCID: PMC7692231.

  4. Irish, J., Blair, S. and Carter, D.A. (2011). The antibacterial activity of honey derived from Australian flora. PLoS One. 6(3): e18229. https://doi.org/10.1371/journal.pone.0018229.

  5. Itoh, S., Yamaguchi, M., Shigeyama, K. and Sakaguchi, I. (2019). The anti-aging potential of extracts from Chaenomeles sinensis. Cosmetics. 6(1): 1. https://doi.org/10.3390/cosmetics6010001.

  6. Jayasakthi, P., Sathya, R., Selvakumar, G. and Chandrasekaran, P. (2025). Assessment on genetic diversity in proso millet (Panicum miliaceum L.) mutants for yield and yield parameters raised during M2 generation. Indian Journal of Agricultural Research. 59(10): 1548-1551. doi: 10.18805/IJARe.A-6425.

  7. Kwok, T., Kirkpatrick, G., Yusof, H., Portokalakis, I., Nigam, P. and Owusu-Apenten, R. (2016). Rapid colorimetric deter- mination of methylglyoxal equivalents for manuka honey. Journal of Advances in Biology and Biotechnology. 7: 1-6. https://doi.org/10.9734/jabb/2016/26592.

  8. Majtan, J., Klaudiny, J., Bohova, J., Kohutova, L., Dzurova, M., Sediva, M., Bartosova, M. and Majtan, V. (2012). Methylglyoxal- induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications. Fitoterapia. 83: 671-677. https://doi.org/10.10 16/j.fitote.2012.02.002.

  9. Pappalardo, M., Pappalardo, L. and Brooks, P. (2016). Rapid and Reliable HPLC Method for the Simultaneous Determination of Dihydroxyacetone, Methylglyoxal and 5-Hydroxymethy- lfurfural in Leptospermum Honeys. PLoS One. 11(12): e0167006. https://doi.org/10.1371/journal.pone.0167006. 

  10. Saikaly, S.K. and Khachemoune, A. (2017). Honey and Wound Healing: An Update. American Journal of Clinical Dermatology. 18: 237-251. https://doi.org/10.1007/s40257- 016-0247-8.

  11. Sandhu, S.J., Parida, A. and Hegde, S. (2025). Advances in anaesthesia and analgesia for laboratory animals- current practices and future directions: A review. Indian Journal of Animal Research. 59(10): 1613-1620. doi: 10.18805/IJAR.B-5593.

  12. Saranraj, P., Sivasakthi, S. and Feliciano, G.D. (2016). Pharmacology of honey: A review. Advances in Biological Research. 10: 271-289. https://doi.org/10.5829/idosi.abr.2016.10.4.104104.

  13. Stephens, J.M., Schlothauer, R.C., Morris, B.D., Yang, D., Fearnley, L., Greenwood, D.R. and Loomes, K.M. (2010). Phenolic compounds and methylglyoxal in some New Zealand manuka and kanuka honeys. Food Chemistry. 120: 78- 86. https://doi.org/10.1016/j.foodchem.2009.09.074.
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    Full Research Article

    Quantification of Methylglyoxal in Honey using LC-MS Method

    K
    K. Sonia1,*
    M
    M.A. Suman Raj1
    P
    P. Dharshini Priya1
    S
    S. Sabarish1
    R
    R. Kaviya1
    S
    S. Sayalee1
    S
    S. Sayalee1
    1Department of Pharmaceutical Chemistry, Sri Ramachandra Faculty of Pharmacy, SRIHER(DU), Porur, Chennai Chennai-600 116, Tamil Nadu, India.

    ABSTRACT

    Background: Honey is sweet and viscous substance produced mainly by honey bees. It contains simple sugars like glucose and fructose, making it sweet and easily digestible.

    Methods: The present study aimed to provides a comprehensive and critical review of the different types of adulteration, common sugar adulterants and detection methods and draws a clear perspective toward the impact of honey adulteration on human health.

    Result: The MGO can be identified and quantified in different honey samples within the concentration range of 50 mg to 600 mg/kg. This study investigated the release pattern of MGO from five honey-based (Lepto- spermum spp.) commercial products and the honey matrix itself. The conversion of MGO into the more stable compound 2-methylquinoxaline allows for an easy evaluation of the MGO content. The recovery study showed a detectable MGO amount of around 90%. The correlated MGO amount in different samples of honey was calculated accordingly. Overall, there is no release pattern for MGO was noticed from the honey matrix as well as from all investigated commercial products.

    INTRODUCTION

    Honey is identified as a pleasant and fresh edibles so, feasible absorbed as a sweetener in addition to as medicine due to its curative effect on human health (Atrott et al., 2012). It is vulnerable  to adulterants give rise to by humans that operate the quality of honey (Saranraj et al., 2016). Despite the fact that honey consumption has significantly expanded globally in new years, its safety is not routinely evaluated and tracked (Irish et al., 2011). Their expectation and significance in this high quality  product has decreased. Since the sum total of consumers of honey have enlarged in new years, (Itoh et al., 2019). Honey debasement are any material that are added to the pure honey in Fig 1 (Fakhlaei et al., 2020) In this sense, this study offers a thorough and critical analysis of the many forms of defilement, typical sugar adulterants and detection techniques. It also presents a clear account of the consequence of honey adulteration on human health (Kwok et al., 2016). Adulteration causes the consumer’s blood sugar levels to rise, whatever can lead to hypertension, diabetes, obesity and abdominal increase in body mass (Majtan et al., 2012 and Pappalardo et al., 2016).

    Fig 1: Different brands of honey sample.


           
    According to multiple in vivo research designs, the liver is most commonly affected by honey adulterants, followed by the kidney, heart and brain (Saikaly et al., 2017).
           
    Methylglyoxal (MG) (Fig 2) is created by the disintegrating of glycolytic intermediates, by lipid peroxidation systems, in the oxidation of acetone and the catabolism of amino acids. As a highly reactive α-oxoaldehyde, it has strong interactions with proteins and nucleic acids., causing mutations and apoptotic cell death, or generating Advanced glycation end products (AGE) that take part in the patho-physiology of issues related to diabetes as well as the develop-ment of a number of age-related illnesses, cardio vascular disease, malignant and Alzheimer’s disease, cancer (Stephens et al., 2010). The aim of the analysis is to quantify methylglyoxal present  in different brands of honey available in India, using LC-MS Method and plan of work shown in Fig 3.

    Fig 2: Structure of methyl glyoxal.



    Fig 3: Plan of work.

    MATERIALS AND METHODS

    Materials
     
    The different brands of Honey were procured from the   local supermarket.  The Methyl glyoxal were purchased from MP Biologicals. The instrument used is Digital Balance - Sartorius group.
     
    Preparation of buffer
     
    Add 200 ml of water, to 500 ml of volumetric flask, 1 ml of formic acid and made volume up with HPLC grade water.

    Mobile phase preparation
     
    Before use of mobile phase, 20 ml of buffer, 80 ml of HPLC-grade acetonitrile and methanol in a 20:80 v/v ratio were combined and shaken.

    Preparation of Standard
     
    Weighed amounts of 2 mg for each MGO was dissolved in methanol; further dilutions are made in Methanol: Water (50:50% V/V).
     
    Standard stock preparation in diluents
     
    Methanol: water (HPLC grade) (50: 50% v/v).

    Sample extraction procedure
     
    500 µL of the Honey samples were used for sample preparation procedure. To this sample 500 µL of Phenylene diamine reagent was added. Mixture was vortexed well for few seconds and sample kept in surrounding temperature for 60 mins for completion of reaction. From above reaction mixture 50 µL of above mixture was taken and mixed to 1.5 mL of methanol and then vortexed well. Transferred the sample in to injector vial for UFLCMS analysis. The study  was bring out using UFLC-MS/MS.
     
    Chromatographic conditions
     
    The temperature of the auto sampler retain  at 40oC.Before being utilized, the mobile phase remained ultrasonically degassed subsequently going all the time a 0.22 m Millipore membrane filter. LC operated for six minutes while the column oven 40oC was retained. HPLC-grade water and acetonitrile made up the rinse solution. (80:20, v/v) (Sravani et al., 2024).
           
    API 4000 UFLC-MS/MS equipment were pre-ownfor the analysis.(Linear triple quadrupole Mass Spectrometer), which was equipped with an auto-injector. The Analyst 1.6.3 program managed the mass data collection and processing and the specifics are given as follows in (Table 1). The operating conditions for the mass spectrometer in positive mode were as mentioned in (Table 2).

    Table 1: LC-MS conditions triple quadrupole LC/MS/MS mass spectrometer.



    Table 2: LC-MS chromatographic conditions for methyl glyoxal.



    Development and optimization of the LC method
     
    The mobile phase was altered to provide adequate selectivity and sensitivity in order to achieve the desired chromatographic conditions. in a short dissociation time. Fig 4 to 11 display the results of the standard solution (Sandhu et al., 2025 and Jayasakthi et al., 2025). Methanol was used to increase peak symmetry and save analysis time. Columns from various sources were assessed and the Inertsil ODS C18, 150 mm, 4.6 mm and 5 µm analytical column was selected, because it produced appropriate peak properties, such as tailing factor and the best chromatographic performance. Moreover, acceptable resolution of MGO was applied, verifying the proposed method’s capacity to demonstrate stability. An appropriate peak symmetry and a steady baseline and a reasonable separation were attained with mL/min. It is tabulated in Table 3 and Fig 12 to 16.

    Table 3: Quantitate Mode of the standard and Sample



    Fig 4: Chromatogram of standard A.



    Fig 5: Chromatogram of Standard B.



    Fig 6: Chromatogram of standard C.



    Fig 7: Chromatogram of standard D.



    Fig 8: Chromatogram of standard E.



    Fig 9: Chromatogram of Standard F.



    Fig 10: Chromatogram of standard G.



    Fig 11: Chromatogram of standard H.



    Fig 12: Chromatogram of annai honey sample.



    Fig 13: Chromatogram of apis himalaya honey sample.



    Fig 14: Chromatogram of kodaikannal honey sample.



    Fig 15: Chromatogram of lion kashmir honey sample.



    Fig 16: Chromatogram of saffola honey sample.

    RESULTS AND DISCUSSION

    The Analyst 1.6.3 program managed the mass data’s acquisition and processing; the specifics are listed in Table 1. The mass spectrometer was operating in positive mode under the settings listed in Table 2. The proposed method’s capacity to demonstrate stability was confirmed by the achievement of an appropriate resolution of MGO. Using mL/min, a good separation with a stable baseline and good peak symmetry was obtained and tabulated in Table 3. (Sravani et al., 2024).

    CONCLUSION

    The MGO in different honey samples was found to be within the range of 50 mg to 600 mg/kg concentration. This study examined the MGO release pattern from five commercial honey-based (Lepto-spermum spp.) products as well as the honey matrix. The MGO in the sample can be easy to evaluate because it can be changed into the more stable compound 2-methylquinoxaline. The recovery study found that the MGO level was about 90% detectable. The linked MGO content in various honey samples was computed effectively. Despite the supplier’s failure to establish any evidence of MGO concentration. Overall, neither the honey matrix nor any of the commercial goods under investigation showed any MGO release patterns. Although it does not seem to have an effect on the total amount of MGO released, it was shown that the honey matrix influences the rate of release in addition to the potential impact of formulation excipients on MGO.
           
    The analysis of MGO was difficult and complicated food matrix, such as honey, which was explained. In conclusion, methylglyoxal can be rapidly and simply quantified using UFLC-MS. This application highlights the key advantages of the method, including rapid sample preparation, strong tolerance to complex matrices and high throughput.

    ACKNOWLEDGEMENT

    The present study was supported by Sri Ramachandra Faculty of Pharmacy, SRIHER(DU).

    Disclaimers
     
    The opinions in this article belong entirely to the authors and do not reflect the official position of their affiliated organizations. While the authors aim for accuracy, they are not liable for any losses or damages caused by using this information.
     
    Informed consent
     
    There is no animal used for experiments.

    CONFLICT OF INTEREST

    The authors declare no financial or non-financial conflicts of interest. This research was conducted independently without any external sponsorship influencing its findings or publication.

    REFERENCES


    1. Atrott, J., Haberlau, S. and Henle, T. (2012). Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey. Carbohydrate Research. 361: 7-11. https://doi.org/10.1016/j.carres.2012.07.025.

    2. Burns, D.T., Dillon, A., Warren, J. and Walker, M.J. (2018). A Critical review of the factors available for the identification and determination of mβnuka honey. Food Analytical Methods. 11: 1561-1567. https://doi.org/10.1007/s12161- 018-1154-9.

    3. Fakhlaei, R., Selamat, J., Khatib, A., Razis, A.F.A., Sukor, R., Ahmad, S. and Babadi, A.A. (2020). The toxic impact of honey adulteration: A review. Foods. 9(11): 1538. doi: 10. 3390/ foods9111538. PMID: 33114468; PMCID: PMC7692231.

    4. Irish, J., Blair, S. and Carter, D.A. (2011). The antibacterial activity of honey derived from Australian flora. PLoS One. 6(3): e18229. https://doi.org/10.1371/journal.pone.0018229.

    5. Itoh, S., Yamaguchi, M., Shigeyama, K. and Sakaguchi, I. (2019). The anti-aging potential of extracts from Chaenomeles sinensis. Cosmetics. 6(1): 1. https://doi.org/10.3390/cosmetics6010001.

    6. Jayasakthi, P., Sathya, R., Selvakumar, G. and Chandrasekaran, P. (2025). Assessment on genetic diversity in proso millet (Panicum miliaceum L.) mutants for yield and yield parameters raised during M2 generation. Indian Journal of Agricultural Research. 59(10): 1548-1551. doi: 10.18805/IJARe.A-6425.

    7. Kwok, T., Kirkpatrick, G., Yusof, H., Portokalakis, I., Nigam, P. and Owusu-Apenten, R. (2016). Rapid colorimetric deter- mination of methylglyoxal equivalents for manuka honey. Journal of Advances in Biology and Biotechnology. 7: 1-6. https://doi.org/10.9734/jabb/2016/26592.

    8. Majtan, J., Klaudiny, J., Bohova, J., Kohutova, L., Dzurova, M., Sediva, M., Bartosova, M. and Majtan, V. (2012). Methylglyoxal- induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications. Fitoterapia. 83: 671-677. https://doi.org/10.10 16/j.fitote.2012.02.002.

    9. Pappalardo, M., Pappalardo, L. and Brooks, P. (2016). Rapid and Reliable HPLC Method for the Simultaneous Determination of Dihydroxyacetone, Methylglyoxal and 5-Hydroxymethy- lfurfural in Leptospermum Honeys. PLoS One. 11(12): e0167006. https://doi.org/10.1371/journal.pone.0167006. 

    10. Saikaly, S.K. and Khachemoune, A. (2017). Honey and Wound Healing: An Update. American Journal of Clinical Dermatology. 18: 237-251. https://doi.org/10.1007/s40257- 016-0247-8.

    11. Sandhu, S.J., Parida, A. and Hegde, S. (2025). Advances in anaesthesia and analgesia for laboratory animals- current practices and future directions: A review. Indian Journal of Animal Research. 59(10): 1613-1620. doi: 10.18805/IJAR.B-5593.

    12. Saranraj, P., Sivasakthi, S. and Feliciano, G.D. (2016). Pharmacology of honey: A review. Advances in Biological Research. 10: 271-289. https://doi.org/10.5829/idosi.abr.2016.10.4.104104.

    13. Stephens, J.M., Schlothauer, R.C., Morris, B.D., Yang, D., Fearnley, L., Greenwood, D.R. and Loomes, K.M. (2010). Phenolic compounds and methylglyoxal in some New Zealand manuka and kanuka honeys. Food Chemistry. 120: 78- 86. https://doi.org/10.1016/j.foodchem.2009.09.074.
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