EXTERNAL CAVITY-QUANTUM CASCADE LASER SPECTROSCOPY AND CHEMOMETRICS FOR

PROTEIN ANALYSIS IN COW’S MILK

Julia Kuligowski1,2*, Andreas Schwaighofer2, Mirta R. Alcaráz2,3, Máximo Vento1,4, Bernhard Lendl2

1Neonatal Research Group, Health Research Institute La Fe, Valencia, Spain 2Institute for Chemical Technology and Analytics, Vienna University of Technology, Vienna, Austria

3Laboratorio de Desarrollo Analítico y Quimiometría, FBCB, Universidad Nacional del Litoral-CONICET, Santa Fe, Argentina 4Division of Neonatology, University & Polytechnic Hospital La Fe, Valencia, Spain

*e-mail: julia.kuligowski@uv.es

The authors acknowledge the Austrian Research Promotion Agency (FFG) within the K-project imPACts (contract no. 843546) for funding. JK acknowledges a Sara Borrell grant (CD12/00667) from Instituto Carlos III (Spanish Ministry of Economy and Competitiveness).

OBJECTIVE

CONCLUSIONS & OUTLOOK

The determination of total protein content in cow’s milk is a routine application of mid-infrared (IR) transmission spectroscopy. However, the quantitation of protein in this kind

of samples demands laborious and time consuming experimental work. We report an analytical method based on the direct spectroscopic determination of casein (Cas), α-

lactalbumin αLA and β-lactoglobulin βLG in cow milk samples.

1. An EC-QCL setup was applied for the determination of Cas, ɑLA and βLG.

2. The present method supports the direct analysis of samples without sample processing.

3. Background correction employing SBC is used for compensation of the milk matrix.

4. Quantification of proteins in off the shelf cow milk samples was carried out using PLS regression.

5. This high throughout method could potentially be employed as a standard tool for quality control of milk.

PARTIAL LEAST SQUARES REGRESSION MODELS FOR PROTEIN QUANTIFICATION

EC-QCL SETUP BACKGROUND CORRECTION

REFERENCES

1. M.R. Alcaráz, A. Schwaighofer, C. Kristament, G. Ramer, M. Brandstetter, H. Goicoechea, B. Lendl,

Anal Chem, 87, 6980-6987 (2015).

Grating angle

determines

emission

wavelength

Fourier Transform IR (FTIR) vs. EC-QCL Spectroscopy • Emission power: EC-QCL (mW) >> Globar (µW)

• Path length for acqueous solutions: FTIR <10 µm // EC-QCL 38 µm

• Flow-through measurements: not feasable (FTIR) // FIA system (EC-QCL)

• LOD: high protein concentration ~10 mg/ml (FTIR) // ~2.5 mg/ml (EC-QCL)

EC-QCL (Daylight Solutions)

1565 cm-1 – 1730 cm-1

Temp. stabilized

(Peltier-cooled)

EC-QCL Measurements [1] • No sample preparation

• Sample volume: 1 ml

• Acquisition time: 10 min

• Reference spectrum: H2O

Science Based Calibration (SBC) Single spectrum:

Set of spectra:

• Analyte signal: mean analyte spectrum ( ) ± standard dev. ( )

• Spectral noise: mean noise value ( ) ± covariance matrix ( )

If analyte concentration constant, after mean centering, spectra only contain information about the noise!

Concentration

Transposed analyte spectrum

Signal not derived from analyte

(Matrix, instrumental noise)

Pseudo-inverse of

covariance of spectral noise

Matrix components (e.g. sugar, fat) in milk modify background absorption

Variation in the sample’s water content

Background correction necessary

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1. Collect lactose spectrum 2. Collect spectra of protein

standard mixtures

3. Collect spectra of (spiked)

cow milk samples

4. Compute optimum b

vector (bopt)

5. Predict lactose concentration

(ypred) in milk spectra (xpred)

6. Subtraction of lactose

proportion from signal

Mean analyte

concentration

Mean noise

spectrum

Protein concentrations In commercially available milk

samples

• Cas: 24.5±1.4 mg mL-1

• αLA: 1.4±0.9 mg mL-1

• βLG: 2.4±0.2 mg mL-

Recoveries Spiked milk samples

• Cas: 95±8%

• αLA: 101±22%

• βLG: 99±4%

PLS models for protein

determination • Calibration set: spectra of

10 standard mixtures and

their concentrations

• Cross validation: Leave-

one-out

• Validation set: background

corrected spectra of

(spiked) milk samples

0 10 20 300

10

20

30

40

Measured Cas [mg/mL]Pre

dic

ted

Cas

[m

g/m

L]

Casein Preprocessing: Mean Center; Num. LVs: 4

RMSEC: 0.5; RMSECV: 1.3; RMSEP: 2.1

Bias: 0; CV Bias: -0.2

R2 Cal: 0.998; R2 CV: 0.990

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1

1.5

2

2.5

Wavenumber [cm-1]

VIP

Sc

ore

s f

or

Cas

2 4 6 8 10

0

5

10

Measured aLA [mg/mL]

Pre

dic

ted

aL

A [

mg

/mL

]

ɑLA Preprocessing: Mean Center; Num. LVs: 4

RMSEC: 0.3; RMSECV: 0.7; RMSEP: 0.7

Bias: 0; CV Bias: 0.03

R2 Cal: 0.990; R2 CV: 0.94

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1

1.5

2

2.5

Wavenumbers [cm-1]

VIP

Sc

ore

s f

or

aL

A

VIP Scores for Cas

Significance Threshold

Cas 20 mg/mL x 200

βLG Preprocessing: 1st Derivative (order: 3, window: 15 pt), Mean

Center; Num. LVs: 4

RMSEC: 0.10; RMSECV: 0.3; RMSEP: 0.20

Bias: 0; CV Bias: -0.0011

R2 Cal: 0.9990; R2 CV: 0.990

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2

3

4

Variable

VIP

Sc

ore

s f

or

Y 3

0 5 100

5

10

Measured bLG [mg/mL]

Pre

dic

ted

bL

G [

mg

/mL

]

VIP Scores for aLA

Significance Threshold

aLA 20 mg/mL x 200

Fit

1:1

Calibration

Test

VIP Scores for bLG

Significance Threshold

bLG 20 mg/mL x 200

Fit

1:1

Calibration

Test

Fit

1:1

Calibration

Test