Post on 06-May-2018
Improved LC Methods in Food QC and Routine Food Analysis
Dayin Lin, Ph.D.
Credits: Mr. Adam Horkey, Schwabe NA
(USA) Dr. Udo Huber & Dr. Sonja
Schneider (Agilent DE)
Agenda
• Agilent UHPLC for Food QC
• Faster QC with 1290 Infinity LC
• Agilent 1200 Infinity HDR DAD and cost-saving solution
• 1220 Mobile LC for Beer QC
Agilent Food Seminar
2
Agilent Liquid Phase Separations Portfolio Overview
1290 Bin
1260
Bin
1220
Grad
1220
Iso
SFC 1260 SFC and SFC/MS
1260 Hybrid SFC/UHPLC-MS
Bio-inert LC 1260 Bio-inert LC
1260 Bio-SEC
1290 Quat
1260
Quat
1260
Iso
Page 3
Dissolution 708/709, 400, BIO-DIS,
8000, MQ 850
High-end UHPLC
Core UHPLC
HPLC-Chip/MS UHC-NCE, Phospho Peptide
mAB-Glyco, HiP Protein ID
Lowflow LC 1290 Micro, 1260 Cap
1260 Nano
GPC-SEC 1260 GPC, 1260 MDS,
GPC 50, GPC 220
Cap. Electrophoresis 7100 CE and CE/MS
Workflow Automation Solutions Multi-method/Walk-up, Method Dev., ISET, 2D-LC, Online SPE, HT LC/MS, ACR,
Online Enrichment and Clean-up, HDR-DAD, Mobile LC, Online Sampling, etc
Preparative LC 1260 uS, 1260 AS, 1260 PS
218, SD1
HDR
Agilent Food Seminar
Analysis of Curcumins with UHPLC
1290 Infinity
Binary LC
Abstract: Improving traditional USP method in analysis of curcuminoids
with Agilent 1290 Infinity LC and STM column
Food Testing
Pub. number: 5991-3340EN
Agilent 1290 Infinity LC Agilent’s leading UHPLC
Requirement/benefit:
Significantly improvement over
current USP analysis method for
curcumins: much faster and huge
savings in labor and solvent use.
Agilent Food Seminar
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World of Turmeric Spice
• Natural extract from the Turmeric spice
• Various known benefits
• Arthritis, digestive problems and urinary issues
• Low energy
• Variety of skin and wound conditions
• Alzheimer’s disease, diabetes, allergies
• USP method for Analysis
Agilent Food Seminar
5
Current USP Method
Curcuminoids in turmeric extract using USP method with an Agilent
ZORBAX Eclipse Plus C18, 4.6x250 mm, 5µm column
USP Curcuminoids RS
Lot F0H161 (Cat. 1151866)
USP Monograph: Curcuminoids (PF 33 (6))
Test: Content of curcuminoids
Solution: Standard Solution 1
Agilent Food Seminar
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Agilent 1290 Infinity LC Method with STM column
Curcuminoids in turmeric extract using an Agilent 1290 Infinity LC with
an Agilent ZORBAX Eclipse Plus C18, 2.1x50 mm, 1.8 µm column
Agilent Food Seminar
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Summary of Improvements - 1290 LC method vs. USP method
• 30x faster with Agilent 1290 Infinity LC method
• Similar peak resolution, much higher sensitivity
• 95% reduction in solvent consumption
• Significant cost saving in labor
Agilent Food Seminar
9
QA/QC of Vitamin Tablets
1290 Infinity
Binary LC
Preliminary: Analysis of high-dosed and low-dosed vitamins in one run
using the Agilent Infinity High Dynamic Range DAD
Food Testing & Agriculture
Pub. number: 5991-1737EN
Agilent 1200 Infinity Series High Dynamic Range (HDR) DAD
solution 30x Wider Linear Range
Requirement/benefit:
Quantitation of high abundant
(e.g. vit. C) and low abundant
vitamins (e.g. vit. B12) for the
QA/QC of the tablets in a single
run. Saves time, solvent, waste
and therefore money for the
customer.
Agilent Food Seminar
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1.4 1.8 2.0 1.3
>10
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
1040 > 1090 1090 > 1050 1050 > 1100 1100 > 1200 1200 >1290
History of DAD Sensitivity Gain The last 30 years
Detector Model (Intro Year)
Se
nsitiv
ity G
ain
(1988) (1986) (1995) (2006) (2010)
1260 / 1290 Infinity
Diode Array Detector
Agilent Food Seminar
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History of DAD Linearity Gain The last 30 years
Detector Model (Intro Year)
1.4 1.7 2.0 1.4 2.9
> 30
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
1040 to 1090 1090 to 1050 1050 to 1100 1110 to 1200 1200 to 1290/60 1200 to HDR
Lin
ea
rity
Ga
in
(1988) (1986) (1995) (2006) (2010) (2011)
1200 Infinity HDR-DAD
With HDR
>30X Linearity
Agilent Food Seminar
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Deuterium
Lamp
Max-Light Cartridge Cell
10 mm or 60 mm path length
Programmable
or fixed slit
1024 element
diode-array
Grating Mirror
Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber
1260 / 1290 Infinity DAD
Agilent Food Seminar
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Effects of path length increase
Peak Dispersion
60 mm pathlength
large geom. cell volume (78 µL)
lower light transmission
- Loss of resolution
- Loss of signal height
10 mm pathlength
small geom. cell volume (13 µL)
high light transmission
Conventional flow cells:
60 mm pathlength
4 µL σV dispersion volume
Max-Light High Sensitivity cell:
Optofluidic waveguides
(total internal reflection)
Agilent Food Seminar
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10x Higher Sensitivity 1290/1260 Infinity DAD compare to 1200 Series DAD SL
1260/1290 DAD
60 mm
1200 DAD SL
10mm
Height [mAU] 28.876 4.938
Noise [mAU] 0.0098 0.0190
Signal/ noise 2944 259
Increase 11.4 x
min 0 1 2 3 4 5
mAU
0
5
10
15
20
25
30
Columns: 150 x 4.6mm Zorbax SB C18, 5µm
Sample: Anthracene: 835 pg/µL
Mobile phase: A: Water, B: Acetonitrile
Elution: isocratic 80 % B
Injection volume: 5 µL
Flow: 1.5 mL/min
DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm
1200 Series
DAD / DAD SL
1260/1290
Infinity DAD
Agilent Food Seminar
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Linearity, linear range and dynamic range 1200 Series Diode Array Detector, 10 mm flow cell
Conc [µg/mL]
Detector Signal
[AU]
LOD
(3x S/N) Conc (upper limit)
linear range
Detector
noise
7*10-6
Conc (upper limit)
dynamic range
Linear range
Dynamic range
2.0
Slope = Response Factor
(Sensitivity)
HDR- DAD
HDR- DAD
Upper limit
5
10_3
__
NoiseDetector
LimitUpperrangeLinear
10 mm
Agilent Food Seminar
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30x Wider Linear Range with HDR-DAD 3.7 mm and 60 mm and Max-Light flow cell
60 mm path-length
3.7 mm path-length
Computing the signals from 60 mm path-length for the low concentration
3.7 mm path-length for the high concentration
Detector Signal
[AU/cm]
Conc
[µg/mL]
Agilent Food Seminar
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30x Wider Linear Range with HDR-DAD 3.7 mm and 60 mm and Max-Light flow cell
Conc
[µg/mL]
Detector Signal
[AU/cm]
10 x higher sensitivity ~3x higher upper limit
30x lower Limit of Detection (LOD)
30x lower Limit of Detection (LOD) for impurity analysis
- for more reliable automated peak integration
- higher area precision
60 mm
3.7 mm
Agilent Food Seminar
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1290/1260 High Dynamic Range – HDR Increasing Linear UV Range by >30x
Technology Cluster of two DADs
DAD 1: 60 mm Cell – for low
concentrations
DAD 2: 3.7 mm Cell – for high
concentrations
Output: One, combined signal,
normalized to 10 mm path length (HDR
range: 0.6 x 10-6 to 6.7 AU/cm) vs for
1200 Series: 7 x 10-6 to 2 AU/cm)
Control / Usability Like standard 1290/1260 DAD: Control,
data analysis, reporting
Investment Protection Existing 1290/1260 DAD can be upgraded
(by 2nd DAD)
Existing 1100/1200 Systems can be
upgraded
(by two 1290 or 1260 DAD) 3.7mm
60mm
Clustered DAD
DAD 1:
DAD 2:
Agilent Food Seminar
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QA/QC of Vitamin Tablets Scope
• DAD analysis of main and trace components might require two separate
analyses to be able to quantify all components.
• This can be solved by using the 1200 Infinity HDR DAD solution by the
enhanced dynamic range.
• In vitamin tablets some vitamins are present at high concentrations (e.g. vit. C),
whereas for example vitamin B 12 is present in very low concentrations.
• In the literature for vitamin B12 different sample preparation procedures and
different chromatographic methods are used to be able to detect and quantify
vitamin B 12 in vitamin preparations.1,2
• Until now it was not possible by applying one sample preparation and one
chromatographic method to detect and quantify all water soluble vitamins
present in in a vitamin tablet.
1 U.Kirchner, K.Degenhardt, G.Raffler, Journal of AOAC International Vol.95, No4, 2012 2 Rada Amidzic et al, J.Serb.Chem.Soc.70 (10) 1229-1235 (2005)
Agilent Food Seminar
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QA/QC of Vitamin Tablets Chromatogram
No: Name:
1 Vitamin C (B)
2 Niacinamid (B)
3 Pantothenic acid (A)
4 Vitamin B6 (A)
5 Vitamin B1 (B)
6 Vitamin B12 (C)
7 Vitamin B2 (C)
Column ZORBAX RRHT Eclipse plus C18, 4.6 x 150 mm, 1.8 µm
Mobile phase A A=Water + 4 g/L KH2HPO4 + 0.5 g/L hexanesulfonate, pH 3 with phosphoric acid
Mobile phase B Methanol
Gradient at 0 min 3%B, at 1 min 3%B, at 10 min 34%B, at 11 min 50%B
Flow rate 1.2 mL/min
Col.temp. 40 °C
Inj. volume 2 µL
Data acq. (DAD) A: 220/10 nm, B: 265/10 nm, C: 360/10 nm (ref. off)
min 2 4 6 8 10 12
mAU
0
500
1000
1500
2000
1
2
3
4 5
6
7
Agilent Food Seminar
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QA/QC of Vitamin Tablets 1260 Infinity DAD with 10 mm path length cell
min 2 4 6 8 10 12
mAU
0
500
1000
1500
2000
>2000 mAU linearity limit
Red: 220 nm
Blue: 265 nm
Green: 360 nm
min 10 11 12
mAU
0
0.1
0.2
0.3
0.4
0.5
0.6
Peak height = 0.145998 mAU
PtoP noise = 0.03282 mAU
S/N = 4.4 Vit.
B1
2
Agilent Food Seminar
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QA/QC of Vitamin Tablets 1260 Infinity DAD with 60 mm path length cell
min 2 4 6 8 10 12
mAU
0
500
1000
1500
2000
2500
3000
3500
4000
>350 mAU linearity limit
min 10 10.2 10.4 10.6 10.8 11
mAU
0
0.2
0.4
0.6
0.8
Peak height = 0.768691 mAU
PtoP noise = 0.04776 mAU
S/N = 16.1
Vit.
B1
2
Agilent Food Seminar
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QA/QC of Vitamin Tablets 1260 Infinity HDR DAD
min 0 2 4 6 8 10 12
mAU/cm
0
250
500
750
1000
1250
1500
1750
2000
>5400 mAU linearity limit
Typically 6700 mAU
min 9.5 10 10.5 11
mAU/cm
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16 Peak height = 0.126319 mAU/cm
PtoP noise = 0.007922 mAU/cm
S/N = 16
Vit.
B1
2
Agilent Food Seminar
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QA/QC of Vitamin Tablets Summary of results
Compound
Quantitation with
1260 Infinity DAD
with 10 mm cell
Quantitation with
1260 Infinity DAD
with 60 mm cell
Quantitation with
1260 Infinity DAD
HDR
Vitamin C
Niacinamid
Pantothenic acid
Vitamin B6
Vitamin B1
Vitamin B12
Vitamin B2
Agilent Food Seminar
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QA/QC of Vitamin Tablets Summary of results
• The analysis of vitamin B 12 and other water soluble
vitamins present in vitamin tablets is possible in a single
run using the Agilent 1200 Infinity HDR DAD
• 200 pg (injected amount) of vitamin B12 can be quantified
• 6000 ng (injected amount) of vitamin C can be quantified
simultaneously
• Tedious sample preparation procedures for vitamin B 12
can be avoided
Agilent Food Seminar
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Onsite QC of Beers
1220 Infinity
Mobile LC
Quantification of Isohumulones and Reduced Isohumulones Using the
Agilent 1220 Infinity Mobile LC Solution
Pub. number: 5991-3510EN
Agilent 1220 Infinity Mobile LC Now the LC is near your sample!
Requirement/benefit:
With the mobile LC the analysis can
be easily done at the site that
produces beers.
Food Testing
Agilent Food Seminar
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Mobile Beer Testing is Now Possible
• Analysis of isohumulones in different beer samples
• Direct injection of beer samples and isocratic elution mode enables the
user to perform easy on-site measurements in a mobile laboratory
• With this setup, nonreduced and reduced isohumulone standards were
analyzed with high precision and linearity
• Isohumulones were determined and quantified in 14 different beer
samples
• International Bitterness Units (IBU) were calculated for the tested beer
samples revealing significant differences from, for example, lager beer to
pils
• Reduced isohumulones could only be detected in one beer sample, an
American lager beer
28
Agilent Food Seminar
Chromatographic Conditions
29
Solvent
Flow rate
Stoptime
Injection volume
Temperature TCC
DAD
Peak width
ACN/H2O + H3PO4 to pH 2.8 (52:48, v/v) +
1mL EDTA 0.1M per liter solvent
1.8 mL/min
20 min
5 (standards) or 20 µl (beer samples)
injection with needle wash
35°C
270 nm/ 4 nm, Ref.: OFF
>0.025 min (0.5 s response time) (10 Hz)
Agilent Food Seminar
One Step Separation
30
1
2
3
4
5
6
7
9
8
Nr. Substance
1 Trans-isocohumulone
2 Trans-isohumulone
3 Trans-isoadhumulone.
4 Tetrahydro-trans-isocohumulone
5 Tetrahydro-cis-isocohumulone
6 Tetrahydro-trans-isohumulones
7 Tetrahydro-cis-isohumulones
8 Tetrahydro-itrans-soadhumulone
9 Tetrahydro-cis-isoadhumulone
min 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
300
Separation of nine isohumulone standards, nonreduced (Peaks 1–3) and
reduced (Peaks 4–9) with isocratic elution
Agilent Food Seminar
Linearity and precision in RSD (%) for all nine analyzed iso-α-acids
31
Nr. Iso-α-acids
Correlation
coefiicient
R
RT
RSD
[%]
Area
RSD
[%]
1 Trans-isocohumulone 0.99987 0.097 0.199
2 Trans-isohumulone 0.99987 0.083 0.157
3 Trans-isoadhumulone. 0.99988 0.099 0.158
4 Tetrahydro-trans-isocohumulone 0.99989 0.102 0.093
5 Tetrahydro-cis-isocohumulone 0.99988 0.065 0.148
6 Tetrahydro-trans-isohumulones 0.99989 0.092 0.142
7 Tetrahydro-cis-isohumulones 0.99987 0.062 0.148
8 Tetrahydro-itrans-soadhumulone 0.99957 0.089 0.693
9 Tetrahydro-cis-isoadhumulone 0.99989 0.064 0.145
Agilent Food Seminar
Comparison of IBUs, literature vs. experimental
32
Yeast type Beer type IBU experimental IBU literature*
top-fermented
(Saccharomyces cerevisiae)
Weizen 11 8-15
Kölsch 23 20-30
Irish Stout 40 30-45
Northern English Brown
Ale 24 20-30
bottom-fermented
(Saccharomyces
carlsbergensis)
Premium Lager 26 18-30
American Premium Lager 19
Lager 14 8-15
Export 27 23-30
Bock 29 23-35
Pils 26
25-45, partly up to
100
Pils 38
Pils 27
Pils 60
Pils, alcohol-free 49
*The literature IBUs were taken from the Beer Judge Certification Program, Inc.
Agilent Food Seminar
Separation of isohumulones (A) and of reduced ones (B)
33
min 2 4 6 8 10 12 14 16 18
mAU
0
2
4
6
8
1
2
3
4
5 6
1 Trans-tetrahydroisocohumulone
2 Cis-tetrahydroisocohumulone
3 Trans-tetrahydroisohumulone
4 Cis-tetrahydroisohumulone
5 Trans-tetrahydroisoadhumulone.
6 Cis-tetrahydroisoadhumulone.
Be
er
ma
trix
min 2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
1
2
3
4
5
6
1 Trans-isocohumulone
2 Cis-isocohumulone
3 Trans-isohumulone
4 Cis-isohumulone
5 Trans-isoadhumulone.
6 Cis-isoadhumulone.
Be
er
ma
trix
A
B
Agilent Food Seminar
Summary of Beer Bitterness Analysis
34
• Easy analysis with direct injection (without SPE) and
isocratic elution
• Analysis of nonreduced and reduced isohumulones was
highly precise and linear with correlation coefficients over
0.999 %.
• The IBUs were calculated, and, as expected, significant
differences were found from weizen beer to pils
• Nonreduced isohumulones were detected in most beers
except for the American premium lager, which contained
only reduced isohumulones.
• The 1220 Infinity Mobile LC Solution is a robust and rugged
system that enables easy on-site measurement of
isohumulones in beer in a simple analytical setup
Agilent Food Seminar