Analysis of Lactose in Milk by HPLC-RI using Chromolith^® NH2 Column

INTRODUCTION

Lactose also referred to as milk sugar (Figure 1) is the principal dietary disaccharide component found in milk. The analysis of Lactose in milk is important as it contributes to the sensory and functional properties of milk (1). A considerable number of methods have been developed for the determination of carbohydrates in milk, including older and less sensitive ones, such as gravimetric, polarimetric, enzymatic, or spectrophotometric analysis. Further, the Association of Official Analytical Chemists (AOAC) Method 984.15 utilizes enzymatic hydrolysis of lactose to glucose and galactose at pH 6.6 by β-galactosidase (2). This method is time-consuming and demands extensive reagent preparations.

HPLC is the most popular method of choice to analyze a diverse group of compounds because of its simplicity, speed, and accuracy. Furthermore, the monolithic silica technology of Chromolith^® columns enable reliable results with extended column lifetime and reduced need for sample preparation for matrix-rich samples due to its high matrix tolerance. Hence, we demonstrate an application for the determination of Lactose in milk by HPLC-RI using Chromolith^® NH2 column for system suitability, linearity, repeatability, percentage recovery, LOD, and LOQ.

Figure 1. Chemical Structure of Lactose

EXPERIMENTAL CONDITIONS FOR THE ANALYSIS OF LACTOSE

HPLC Conditions
Column:	Chromolith® NH2 column, 100 x 4.6 mm
Mobile phase:	Acetonitrile:Water (75:25) v/v, isocratic
Flow rate:	0.5 mL/min
Back pressure:	24 bar (348 psi)
Column temp.:	40 oC
Detector:	RI
Injection:	10 µL
Diluent:	Acetonitrile:Water (50:50) v/v
Standard:	Weigh and transfer Lactose monohydrate standard equivalent to 50 mg to a 10 mL volumetric flask. Add diluent with intermittent swirling, sonicate this solution for 2 min and top up to mark with the diluent. Standard solutions need to be freshly prepared.
Sample:	Take 0.5 mL of sample into a 10 mL volumetric flask. Add acetonitrile: water (70:30) v/v with intermittent swirling and sonicate this solution for about 10 min. Mix well and filter 1 mL of supernatant using a 0.45 µm PVDF syringe filter for HPLC analysis.

CHROMATOGRAPHIC DATA OF LACTOSE ANALYSIS IN MILK

Figure 2. Chromatograms of a) Lactose reference STD solution; b) Lactose in the Cow milk sample

Table 1. System Suitability Data of STD Lactose, 5000 µg/mL

#	Compound	Retention time (min)	Area	Tailing factor
1.	Lactose	8.4	753766	0.9

Table 2. Repeatability of STD Lactose, 5000 µg/mL

Identity	Peak Area
STD 1	753766
STD 2	763284
STD 3	748593
STD 4	753312
STD 5	753766
Mean	744544
Standard Deviation	4786
RSD (%)	0.63

Table 3. Linearity of STD Lactose

Concentration (µg/mL)	Mean Area
50	7575
250	37877
500	64202
1250	180883
2500	339642
3750	564850
5000	763284
6250	939279
7500	1179356

Figure 3. Linearity plot of Lactose reference standard

Table 4. Percentage recovery of Lactose in Milk

No.	Spike Level	µg/mL added	µg/mL found	% Recovery
1	50%	2500	2475	99
2	100%	5000	4960	99.2
3	150%	7500	7448	99.3

RESULTS AND DISCUSSION

An isocratic HPLC-RI method for the determination of Lactose in milk was developed using Chromolith^® NH2 column. The experimental data indicated excellent linearity with an r² value of 0.9989 (Figure 3) for the selected concentration range (50– 7500 µg/mL, Table 3). The LOD and LOQ values were estimated as 325 µg/mL and 987 µg/mL, respectively. The method precision for the assay was below 1% RSD and the percentage recovery was found to be ranging from 99 -99.3% (Table 4).

In conclusion, the data for linearity, system suitability, and repeatability of the method suggest its aptness for the assay of Lactose in milk using Chromolith^® NH2 column.

References

1. Magan JB, O?Callaghan TF, Kelly AL, McCarthy NA. 2021. Compositional and functional properties of milk and dairy products derived from cows fed pasture or concentrate-based diets. Comprehensive Reviews in Food Science and Food Safety. 20(3):2769-2800. https://doi.org/10.1111/1541-4337.12751

2. Official Method 984.15. Lactose in Milk. Enzymatic Method. In Official Methods of Analysis of AOAC International; AOAC: Washington, DC, USA, 2005.