Quality testing for infant formula

Fast and reliable low level lactose determination with IC-PAD

Application Note AN-P-088

Lactose, a disaccharide made of glucose and

galactose, is present in milk and milk products. As the

major carbohydrate in human milk (55–70 g/L), it is

also a main component of infant formulas with

minimum recommendations of 4.5 g/100 kcal [1].

Analytically, such high concentrations can easily be

determined by multi-component methods such as ISO

22184 or lower sensitivity methods like refractive

index detection. In some cases, infants can suffer from

lactose malabsorption. Here, the lactase enzymatic

activity is either limited or absent from birth, impairing

lactose metabolization. The lactose cannot be

hydrolyzed into glucose and galactose in the small

intestine, impeding the absorption of digestible

carbohydrates as monosaccharides. Consequently,

numerous gastrointestinal and extra-intestinal

symptoms and complaints appear (e.g. diarrhea)

[1–3]. Glucose-galactose malabsorption also affects a

very small subset of the population. For all

malabsorption types, it is critical to eliminate any

dietary sources of lactose. Lactose-free nutritional

sources are especially crucial for infants, and these

foodstuffs must properly meet the regulation

requirements (lactose <10 mg/100 kcal) [1,3–5]. To

analyze such low lactose contents in complex

matrices like infant formula, ion chromatography with

pulsed amperometric detection (IC-PAD) offers a

robust and sensitive solution.

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SAMPLES AND SAMPLE PREPARATION

Lactose determination was performed for a broad

spectrum of low lactose sample matrices, comprised

of infant formulas and follow-up baby food such as

the reference materials NIST 2383a (baby food

composite) and NIST 1869 (infant formula powder),

an infant formula from HiPP (HiPP comfort, lactose

reduced), and a commercially available lactose-free

milk (1.5% fat, Spar Switzerland).

Powdered and liquid materials were homogenized

and weighed directly into suitable containers (from

0.1–5 g, 50 mL polypropylene centrifuge tubes). The

sample weight (W

in g) was recorded to the nearest

0.001 g for later calculations. An aqueous extract was

prepared by adding ultrapure water (UPW) to result in

a total volume of 50 mL (W

UPW

in kg). Afterwards,

the vials were capped and mixed vigorously with a

vortex mixer for approximately 20 seconds.

Carrez precipitation is a standard method to remove

proteins and larger molecules from samples in order

to protect the analytical system. Following this

common practice, the reagents were added, and the

final weight noted (W

UPWc

in kg). After thorough

mixing, the samples were centrifuged (5000×g) for 10

minutes and decanted. The covered vials were placed

directly into the autosampler. Increased column

protection can be secured by an additional

ultrafiltration step.

As an alternative, automated sample preparation by

Inline Dialysis with a Low Volume dialysis cell is

recommended. For that, samples were prepared

identically to the aqueous extracts, shaken well, and

covered before placing on the autosampler rack. For

dialysis, no Carrez precipitation is necessary prior to

the analysis, saving time and chemical reagents. Using

the Low Volume dialysis cell requires only 5 mL of

sample.

EXPERIMENTAL

The quantity of lactose in aqueous sample extracts

was determined by ion chromatography (IC) on a

Metrosep Carb 2 - 250/4.0 column using an isocratic

hydroxide eluent (400 mmol/L NaOH) and pulsed

amperometric detection (PAD) with the sweep

waveform (Figure 1).

Together with the Metrohm Thin-Layer amperometric

cell (Au working and Pd reference electrode), a long

electrode lifetime with minimal maintenance is

obtainable. The sweep mode combined with less

turbulent flow in the Thin-Layer cell results in a

smooth baseline, a necessary precondition to analyze

very low concentrations such as in low lactose

products. Flow schemes for direct analysis and

analysis after Metrohm Inline Dialysis are shown in

Figure 2. Although the setup for the dialysis may look

more complex, automation makes the effort

worthwhile for the overall analytical process.

Figure 1. The sweep waveform for carbohydrate oxidation

supports sensitive detection of carbohydrates with a low noise

level.

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RESULTS AND DISCUSSION

Figure 2. Example system configuration for direct lactose analysis (left, sample preparation mandatory as for example Carrez precipitation)

and with optional dialysis (right, no additional sample preparation necessary). Inline Dialysis is optional and can be added to any existing

instrumentation setup. For Carrez precipitation the sample path goes directly from the autosampler into the sample loop (PEEK, 10 μL).

Sample transport occurs by peristaltic pump. From the sample loop, the sample is directly injected onto the column (Metrosep Carb 2 -

250/4.0), where it is eluted with a 400 mmol/L NaOH isocratic eluent before pulsed amperometric detection (PAD).

Lactose elutes in under 20 minutes (Figure 3),

independent of whether direct injection or Inline

Dialysis is used. In contrast to previously published

chromatographic methods, lactose derivatives (i.e.,

epilactose, lactulose, allolactose, and

galactosyllactose), as from prebiotic additives, were

successfully separated from lactose, increasing the

selectivity and accuracy of the method. The overall

working range of the method is 0.05 to 80 mg/L for

liquid lactose standards, with the ability to analyze

samples in a range of 0.2–21,000 mg/100g with

respective dilution. The sample concentrations are

determined from a linear calibration (c(Lactose)

mg/kg) and are calculated based on the sample

weight to give the final lactose content (c(Lactose)

FIN

in g/100 g):

Validation results are shown for infant formula, baby

food, and milk (Table 1). These display the compliance

with the overall acceptance criteria of AOAC for a full

single laboratory validation (RSD

and variability ≤10%

and ≤7%, spike recoveries 85–115% and 90–110%

for an analytical range of 10–100 mg/100 g and >100

mg/100 g, respectively).

The results obtained for the analysis using Carrez

precipitation prior to injection (Table 1) and using

Inline Dialysis were comparable for selected test

samples analyzed as shown in Figure 3. The data give

an excellent verification for using Inline Dialysis as a

time efficient alternative to Carrez precipitation.

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Figure 3. Lactose determination in baby food (NIST 2383a). Comparison of two sample preparation methods: Inline Dialysis (c(lactose) =

51.1 mg/L, orange) and Carrez precipitation (c(lactose) = 49.6 mg/L, blue). The relative standard deviation of the two samples is 2.1%. For

comparison, a 40 mg/L lactose standard (black) is overlaid.

Table 1. Lactose expressed as lactose monohydrate (conversion factor 1.05) in lactose-free infant formula and milk samples determined after

sample preparation by Carrez precipitation. The table shows the repeatability Rr as RSD from individually prepared samples measured within a

short time period (n = 7), the day-to-day variability as RSD over individual prepared samples measured on different days (4–8 d), and spike

recovery as the average of all spike experiments analyzed over several days.

Repeatability (mg/100 g) (RSD

, %)

Day-to-day variability (mg/100g)

(%)

Total spike recovery (%)

NIST 2383a infant food (average 520 ± 27 mg/100 g (n = 7), target 500 ± 100 mg/100 g, recovery 106±6%)

520 ± 27 (5.1) 528 ± 29 (5.5) 103 ± 2

NIST 1869 infant formula powder (average 569 ± 33 mg/100 g (n = 7), target 520 ± 120 mg/100 g, target

recovery 109 ± 6%)

569 ± 33 (5.5) 523 ± 31 (5.9) 98 ± 3

HiPP Comfort infant formula (20.8 g/100 g (n = 7), target 22.1 g/100 g, target recovery 97 ± 4%)

20751 ± 743 (3.6) 22163 ± 258 (1.2) 102 ± 3

Spar aha 1.5% fat, lactose-free milk

2.35 ± 0.18 (7.9) 2.10 ± 0.05 (2.2) 111 ± 2

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CONCLUSION

Infant formula is a very complex and challenging

matrix as it contains all kinds of nutrients (e.g.,

proteins, fats, carbohydrates, vitamins, and minerals).

Dedicated sample preparation and analysis methods

are needed to guarantee high accuracy, sensitivity,

and selectivity for the determination of low lactose

concentrations. IC-PAD overcomes such analytical

challenges with automated inline sample preparation

options and results in excellent separation of lactose

from other matrix components and lactose

derivatives.

The estimated LOQ of 4 mg lactose/100 g in these

sample matrices is lower than the requirements and

known thresholds for lactose-free infant formula

products. Thus, adequate sensitivity and robustness as

shown by long-term analysis and spike experiments is

given by this method. Additionally, the flexibility for

user adapted applications, optional sample

preparation, and/or automation make IC-PAD ideal

for routine analytics and a highly valuable addition to

the laboratory analytical portfolio.

REFERENCES

EFSA. Scientific Opinion: Essential Composition

of Infant and Follow-up Formulae.

Eur. Food

Saf. Auth. EFSA J.

2014,

(7), 3760.

Facioni, M. S.; Raspini, B.; Pivari, F.; Dogliotti, E.;

Cena, H. Nutritional Management of Lactose

Intolerance: The Importance of Diet and Food

Labelling.

J. Transl

Med.

2020,

(1), 260.

EFSA. Scientific Opinion on Lactose Thresholds

in Lactose Intolerance and Galactosaemia.

Eur.

Food Saf. Auth. EFSA J.

2010,

(9), 1777.

European Commission. Comission Delegated

Regulation (EU) 2016/127 of 25 September

2015 Supplementing Regulation (EU) No

609/2013 of the European Parliament and of

the Council as Regards the Specific

Compositional and Information Requirements

for Infant Formula and Follow-on Formula and

as Regards Requirements on Information

Relating to Infant and Young Child Feeding.

Off. J. Eur. Union 251

2016, 29.

FAO/WHO. CODEX Alimentarius - General

Standard for the Labelling of Prepacked Foods

CXS 1-1985.

Int. Food Stand.

Internal reference: AW IC CH6-1435-022021

CONTACT

Metrohm Middle East FZC

B2-21 SAIF Zone, Sharjah,

UAE

120747 Sharjah [email protected]

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CONFIGURATION

940 Professional IC Vario ONE/Prep 1

The 940 Professional IC Vario ONE/Prep 1 is the

intelligent IC instrument without suppression in

combination with Metrohm Inline Sample

Preparation, e.g., Inline Ultrafiltration or Inline

Dialysis. The instrument can be used with any

separation and detection methods.

Typical areas of application:

Anion and cation determinations without

suppression after Inline Ultrafiltration or Inline

Dialysis

UV/VIS applications after Inline Ultrafiltration or

Inline Dialysis

Applications with amperometric detection after

Inline Ultrafiltration or Inline Dialysis

945 Professional Detector Vario – Amperometry

Intelligent stand-alone detector equipped with the IC

Amperometric Detector. Outstanding selectivity due

to the four measuring modes: DC, PAD, flexIPAD, CV.

The excellent signal/noise ratio and the very fast

start-up guarantee the highest in measurement

precision. For use with intelligent IC instruments or as

independent detector.

Metrosep Carb 2 - 250/4.0

The Metrosep Carb 2 - 250/4.0 IC column is

particularly suitable for the determination of

carbohydrates using alkaline eluents and pulsed

amperometric detection. The high-capacity anion

exchanger column is based on a styrene-

divinylbenzene copolymer. It is stable in the range of

pH = 0 - 14 and separates monosaccharides and

disaccharides. It is also suitable for the analysis of

sugar alcohols, anhydrous sugars, amino sugars, etc.

The 250 mm version of the Metrosep Carb 2

separation column is optimized for complex

separations.

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858 Professional Sample Processor – Pump

The 858 Professional Sample Processor – Pump

processes samples from 500 μL to 500 mL. The

sample transfer takes place either with the installed

bidirectional two-channel peristaltic pump or with an

800 Dosino.

IC equipment: Low Volume Inline Dialysis

Accessory set for rapid Inline Dialysis. For use with the

858 Professional Sample Processor and an additional

two-channel peristaltic pump.

IC equipment: Thin-Layer cell: Carb (Au,Pd)

Equipment comprising Thin-Layer cell with additional

accessories, with electrodes for carbohydrate analysis

with a gold working electrode and a Pd reference

electrode.

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MagIC Net 4.0 Professional: 1 license

Professional PC program for controlling all intelligent

Professional IC systems, Compact IC systems and their

peripherals, all detectors and various Auto samplers,

800 Dosino, 771 Compact Interface, etc. The software

permits checks, data acquisition, data evaluation and

data monitoring as well as report generation of ion

chromatographic analyses.

Graphical user interface for routine operations,

extensive database programs, method development,

configuration and manual system control; very

flexible user management, powerful database

operations, extensive data export functions,

individually configurable report generator, control

and monitoring of all system components and the

chromatography results.

MagIC Net Professional complies fully with FDA

Regulation 21 CFR Part 11 as well as GLP.

MagIC Net is available in 16 dialog languages:

German, English, Chinese, Traditional Chinese,

French, Italian, Spanish, Portuguese, Bulgarian, Czech,

Hungarian, Japanese, Korean, Russian, Slovakian,

Polish

1 license

The installation and documents are supplied on a USB

flash drive.