Nutritional Supplements and Cider

Analysis by High Field Superconducting NMR and Benchtop NMR Systems

John C. Edwards, Ph.D.

Process NMR Associates,

84 Patrick Lane, Ste 115, Poughkeepsie, NY 12603

Presented at Marist College – April 1, 2019

www.process-nmr.com www.nmrblog.com Tel: 845-240-1177

NMR Systems
 On-Line Process NMR

45 Systems Installed 1996-2004

Predominantly in Petroleum
and Petrochemical
PUFA Ester 300 MHz 1H NMR Spectra of Samples at Diferent Points in the Process
8

Separator - High DHA

Separator- High EPA

Molecular Distillation

Clathration Raffinate

Clathration Product

First Esterification

Fish Oil

6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4
f1 (ppm)
50 sample initial data set from all points in manufacturing process.
First round of PLS regression analysis revealed concentration outliers that were
linked to limitations in the GC method. This study was performed on improved GC
data values.
Final 50 Sample models were utilized to validate the calibrations on a 24 sample
validation set
 b

p
b

a
Experimental
Varian Mercury - 300 MHz NMR Supercon Spectrometer 300 MHz 1H NMR In chain
4 pulse on pure sample in 5 mm tube, Run Unlocked CH2
In MNova 8.1.2 SPC Files Imported, Stacked, Binned at 3 Hz interval,
Area Normalized to 100, Saved as Transposed Ascii Matrix Ethyl
PLS Regression Performed Thermo Grams IQ and Eigenvector Solo
CH3
EPA-a

EPA-b
DHA w3
CH3

Olefins
w6/ w9
CH3-CH2-O-OC-CH2-R
=C-CH2-C= EtOOC-CH2-R CH3
EPA
R-CH2-C=

DHA

5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
f1 (ppm)
Separator - High DHA

2
DHA EPA
EPA

Separator- High EPA

EPA
EPA

DHA

Comparison of High EPA and High DHA PUFA-Ester after Separation

5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
f1 (ppm)
3 Hz binning interval

0.5 Hz binning interval

 Spinsolve 1H NMR data at 42.5 MHz of a series of fish oils and esterified omega-3 fatty acids
FO-60

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
f1 (ppm)
Model
Linear regression model using Omega-3 EPA Content (wt%) in Fish Oil and
Partial Least Squares calculated with the SIMPLS algorithm
Developed 27-Jul-2014 15:03:05.984
Pharmaceutical Grade Supplements
Author: John@JCEPNA
X-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830
(John@JCEPNA@20140727T140753.60670200 Correlation between 40 second 1H NMR at 42.5 MHz
m:20140727140753.619)
Included: [ 1-55 ] [ 1-830 ]
and primary analysis method – 50 minute GC analysis
6
Preprocessing: Mean Center 3
Y-block: EPA-GC Data.xlsx 55 by 1 5 2
(John@JCEPNA@20140727T141758.52236711

Q Residuals (0.09%)

Y Stdnt Residual 1
m:20140727141758.529) 4 1
Included: [ 1-55 ] [ 1 ]
Preprocessing: Mean Center 3 0
Num. LVs: 8 2 -1
Cross validation: venetian blinds w/ 7 splits
RMSEC: 1.40566 RMSECV: 2.20172 1 -2
Bias: 0 CV Bias: -0.00054578 -3
R^2 Cal: 0.995028 R^2 CV: 0.987838 0
0 10 20 30 40 0 0.2 0.4 0.6 0.8
Hotelling T^2 (99.91%) Leverage
SSQ Table
Percent Variance Captured by Regression Model
80 15
-----X-Block----- -----Y-Block----- 10
60

Scores on LV 2 (2.48%)
Comp This Total This Total
Y CV Predicted 1

---- ------- ------- ------- ------- 5

1 95.78 95.78 84.51 84.51 40
0
2 2.48 98.26 3.47 87.98
20 -5
3 0.29 98.55 9.21 97.19
4 0.76 99.31 0.59 97.78 -10
5 0.43 99.73 0.60 98.38 0
-15
6 0.07 99.80 0.77 99.15
7 0.06 99.86 0.17 99.33 -20 -20
0 20 40 60 80 -100 -50 0 50
8 0.04 99.91 0.18 99.50 Y Measured 1 Scores on LV 1 (95.78%)
Model Omega-3 DHA Content (wt%) in Fish Oil and
Linear regression model using Pharmaceutical Grade Supplements
Partial Least Squares calculated with the SIMPLS
algorithm
Developed 27-Jul-2014 14:50:021.58
Author: John@JCEPNA
Correlation between 40 second 1H NMR at 42.5 MHz
X-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830 and primary analysis method – 50 minute GC analysis
(John@JCEPNA@20140727T140753.60670200
4 4
m:20140727140753.619)
Included: [ 1-55 ] [ 1-830 ]
Preprocessing: Mean Center 3 2

Q Residuals (0.09%)

Y Stdnt Residual 1
Y-block: DHA-GC Data.xlsx 55 by 1
(John@JCEPNA@20140727T140259.39467736
m:20140727140259.401) 2 0
Included: [ 1-55 ] [ 1 ]
Preprocessing: Mean Center
Num. LVs: 8 1 -2
Cross validation: venetian blinds w/ 7 splits
RMSEC: 0.753632 RMSECV: 1.2463
0 -4
Bias: 0 CV Bias: 0.0987959 0 10 20 30 40 0 0.2 0.4 0.6 0.8
R^2 Cal: 0.99605 R^2 CV: 0.989345 Hotelling T^2 (99.91%) Leverage

SSQ Table
Percent Variance Captured by Regression Model 80 20

-----X-Block----- -----Y-Block----- 60

Scores on LV 2 (2.23%)
10
Comp This Total This Total
Y CV Predicted 1

---- ------- ------- ------- ------- 40

1 95.78 95.78 73.08 73.08 0
2 2.23 98.01 6.53 79.61 R^2 = 0.989
20 8 Latent Variables
3 0.55 98.56 15.91 95.53
RMSEC = 0.75363
4 0.50 99.06 1.63 97.16 -10
0 RMSECV = 1.2463
5 0.67 99.73 0.78 97.93 Calibration Bias = 0
6 0.07 99.80 1.42 99.35 CV Bias = 0.098796
7 0.07 99.87 0.15 99.50 -20 -20
0 10 20 30 40 50 60 -100 -50 0 50
8 0.04 99.91 0.10 99.61 Y Measured 1 Scores on LV 1 (95.78%)
Picospin-80 NMR Data – 82.3 MHz
Model
Linear regression model using Omega-3 EPA Content (wt%) in Fish Oil and
Partial Least Squares calculated with the SIMPLS algorithm
Developed 27-Jul-2014 15:03:05.984
Pharmaceutical Grade Supplements
Author: John@JCEPNA
X-block: Final Fish Oil Spectra - 43MHz.csv 55 by 830
(John@JCEPNA@20140727T140753.60670200 Correlation between 40 second 1H NMR at 42.5 MHz
m:20140727140753.619)
Included: [ 1-55 ] [ 1-830 ]
and primary analysis method – 50 minute GC analysis
6
Preprocessing: Mean Center 3
Y-block: EPA-GC Data.xlsx 55 by 1 5 2
(John@JCEPNA@20140727T141758.52236711

Q Residuals (0.09%)

Y Stdnt Residual 1
m:20140727141758.529) 4 1
Included: [ 1-55 ] [ 1 ]
Preprocessing: Mean Center 3 0
Num. LVs: 8 2 -1
Cross validation: venetian blinds w/ 7 splits
RMSEC: 1.40566 RMSECV: 2.20172 1 -2
Bias: 0 CV Bias: -0.00054578 -3
R^2 Cal: 0.995028 R^2 CV: 0.987838 0
0 10 20 30 40 0 0.2 0.4 0.6 0.8
Hotelling T^2 (99.91%) Leverage
SSQ Table
Percent Variance Captured by Regression Model
80 15
-----X-Block----- -----Y-Block----- 10
60

Scores on LV 2 (2.48%)
Comp This Total This Total
Y CV Predicted 1

---- ------- ------- ------- ------- 5

1 95.78 95.78 84.51 84.51 40
0
2 2.48 98.26 3.47 87.98
20 -5
3 0.29 98.55 9.21 97.19
4 0.76 99.31 0.59 97.78 -10
5 0.43 99.73 0.60 98.38 0
-15
6 0.07 99.80 0.77 99.15
7 0.06 99.86 0.17 99.33 -20 -20
0 20 40 60 80 -100 -50 0 50
8 0.04 99.91 0.18 99.50 Y Measured 1 Scores on LV 1 (95.78%)
Model

Linear regression model using

Partial Least Squares calculated with the SIMPLS algorithm
DHA model – 0.02 ppm bin
Developed 15-Nov-2014 23:10:039.19 Picospin 80.3 MHz 1H NMR Data
Author: John@JCEPNA
X-block: Fish Oils - 40 Sample - X Matrix.xlsx 37 by 57 (John@JCEPNA@20141115T230315.72489196
3 4
m:20141115231022.946)
Included: [ 1-11 13-27 29-32 34-40 ] [ 1-2 71-125 ] 2.5 3

Preprocessing: Mean Center 2

Q Residuals (1.12%)

Y Stdnt Residual 1
2
Y-block: DHA.xlsx 37 by 1 (John@JCEPNA@20141115T230330.44825991 m:20141115231022.953) 1
Included: [ 1-11 13-27 29-32 34-40 ] [ 1 ] 1.5
0
Preprocessing: Mean Center 1 -1
Num. LVs: 3
0.5 -2
Cross validation: venetian blinds w/ 6 splits
-3
RMSEC: 1.09711 0
0 5 10 15 20 25 30 0 0.2 0.4 0.6 0.8
RMSECV: 1.20325 Hotelling T^2 (98.88%) Leverage
Bias: 3.55271e-015
CV Bias: 0.0624194 60 20
R^2 Cal: 0.992002
15
R^2 CV: 0.99041 40

Scores on LV 2 (11.40%)
10
SSQ Table Y CV Predicted 1
20 5
Percent Variance Captured by Regression Model
0
0
-----X-Block----- -----Y-Block----- -5
Comp This Total This Total
---- ------- ------- ------- ------- -20 -10
0 10 20 30 40 50 60 -20 -10 0 10 20
1 79.58 79.58 77.43 77.43 Y Measured 1 Scores on LV 1 (79.58%)

2 11.40 90.97 20.16 97.59

3 7.91 98.88 1.61 99.20
 Regression Results for Various NMR Approaches

EPA Content (Wt%) DHA Content (Wt%)

PLS Regression Data R2, SECV (Wt%) R2, SECV (Wt%)
42 MHz NMR 0.988, 2.20 0.989, 1.25
60 MHz NMR 0.993, 2.13 0.992, 1.13
82 MHz NMR 0.984, 2.53 0.990, 1.20
300 MHz NMR 0.988, 1.68 0.991, 1.09
Peak Integral Data 0.981, 2.71 0.964, 2.24
Fused - NMR-FTIR 0.993, 1.62 0.992, 1.08

www.process-nmr.com www.nmrblog.com Tel: 203-744-5905

Question:
Why is this competitors product so good?

Ingredients : Oyster, Barberry,

Dextrose, Snow Lotus, Bombyx Mori L,
Ginger Root, Salfron Crocus.

Sample preparation:
Extract with 20:80 D2O:CD3CN
Centrifuge or Settle
Supernatant analyzed.

Answer:
Also contained ~10 wt% Sulfoaildenafil
A Viagra Analog
 
Typical Herbal Extracts
NaturTech-013-H_FD
Astragalus Root DI Water Extract
9

NaturTech-012-H_FD
Burdock Root Powder H2O extract
8

NaturTech-011-H
Rhubarb Root DI Water extract
7

NaturTech-010-H_FD
Slippery Elm Bark H2O extract
6

NaturTech-009-H_FD
Sheep Sorrel Powder DI Water Extract
5

NaturTech-008-H_FD
Cats Claw Bark H2O extract
4

NaturTech-007-H_FD
Hawthorn Berry DI Water extract
3

NaturTech-006-H_FD
Pau D Arco Inner Bark H2O extract
2

NaturTech-005-H_FD
Chamomile Flower H2O extract
1

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
ControlED-020-H_300 MHz
Control Herbal-ID
20:80 D2O-CD3CN Extract
JCE-PNA-Merc300
CH3
32
31

O S
30 33
13 14 NH 22
18 CH3
12 15 17 21 26
N
23
11 16 N 20
O 19 N
9 24 25
S 5
8
N 4
O 6 27
10
7 N 28
2
3 CH3
1
H3C
29

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
USP-PDE5i-001-H
USP-PDE5i-001-H
Bali Mojo
CD3CN Extract 1H NMR
JCE-PNA-MVX300

USP-027-H
USP-027-H
USP Standard - Tadalafil
1H NMR in CD3CN
JCE-PNA-MVX300

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
PDE5 Inhibitor Screening
43 MHz

60 MHz

300 MHz
43 MHz

60 MHz

300 MHz
43 MHz

60 MHz

300 MHz
43 MHz

60 MHz

300 MHz
Tadalafil Lot 1156-051A1

Acetaminotadalafil Lot 1444-041A5

Thiohomosildenafil Lot 1220-033A10

Hydroxyhomosildenafil Lot 1441-048A6

Vardenafil HCl Lot 1132-035A1

Hydroxyacetildenafil Lot 1233-001A1

Propoxyphenyl Hydroxyhomosildenafil Lot 1427-092A2

Hydroxythiohomosildenafil lot 1222-031A3

Acetildenafil Lot 1218-018A11

Vardenafil HCl Lot 1132-035A1

Sildenafil Citrate Lot 1110-026A1

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
 43 MHz - PDE5i Standards

PDE5i_STD_Sildenafil Citrate

PDE5i_STD_Vardenafil

PDE5i_STD_003-Hydroxyhomosildenafil

PDE5i-STD-004-Acetildenafil

PDE5i-STD-005-hydroxythiohomosildenafil

PDE5i-STD-006-Acetaminotadalafil

PDE5i-STD-007-Propoxyphenylhydroxyhomosildenafil

PDE5i-STD-008-Thiohomosildenafil

PDE5i-STD-009-Tadalafil

PDE5i-STD-010-Hydroxyacetildenafil

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
f1 (ppm)
Sildenafil Standard
Tadalafil Standard

300 MHz – All Male Enhancement Supplements

43 MHz – All Male Enhancement Supplements
USP-PDE5i-003-H
USP-PDE5i-003-H
Maca
CD3CN Extract 1H NMR
JCE-PNA-MVX300

43 MHz

Caffeine
Undeclared Caffeine

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
43 MHz

60 MHz
dehydroepiandrosterone
USP-PDE5i-006-H
USP-PDE5i-006-H
Golden Seal
CD3CN Extract 1H NMR
JCE-PNA-MVX300

43 MHz
Hydrastine

Berberine

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
 USP-PDE5i-007-H
USP-PDE5i-007-H Dehydroepiandrosterone
Extenze DHEA
CD3CN Extract 1H NMR
JCE-PNA-MVX300

43 MHz

Chrysin

Extenze Sexual Response Enhancement 223 mg

Black Pepper extract (fruit)
Cnidium Extract (seed)
Damiana Extract (leaf)
DHEA (98%)
Niacin
Gamma-Aminobutyric Acid (GABA-98%)
Korean Ginseng (root)
Barrenwort Extract (leaf)
L-Arginine HCl (98%)
Licorice Extract (root)
Muira Puama Extract (bark)
Pregnenolone (98%)
Tribulus Extract (fruit)
Folate (Folic Acid)
Yohimbe HCl (98%)
Astragalus Extract (root)13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
f1 (ppm)
Niacin
Tongat Ali Extract (root)
Velvet Bean Extract (seed)
Chrysin (98%)

13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5
f1 (ppm)
 Horny Goat Weed Extract 600 mg
Maca Root Extract 250 mg
Polypodium vulgare Root Powder 25 mg
USP-PDE5i-008-H
USP-PDE5i-008-H
Horny Goat Weed
CD3CN Extract 1H NMR
JCE-PNA-MVX300

43 MHz

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0
f1 (ppm)

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
The Complex Fingerprint Analysis of Cider and Other Alcoholic
Beverages by Quantitative NMR (qNMR) Spectroscopy

John Edwards, Ph.D.

Process NMR Associates, Poughkeepsie

Presented at CiderCon 2018, Baltimore MD

February 1, 2018
 Typical High-End Analysis Performed on Alcoholic Beverages

Good for volatiles Molecules separated by boiling

point and molecular interaction
with coatings present in distillation
column.

Oven used to
temperature ramp
the sample

Liquid Chromatography
Extensive Calibration and
Expensive Standards
Required!!!

Molecules separated by solvent and interaction with column packing material. Different
solvents and columns required to analyze different components – sugars, alcohols, organic
acids often require different tests and instrument set up.
 New Technology - Nuclear Magnetic Resonance (NMR)
Main Chemical Analysis of Molecular Structure Used by Chemists since 1960’s
Technology also Evolved into Magnetic Resonance Imaging during 1970’s
Manufacturers:
NMR Spectrometer Bruker – Germany
JEOL - Japan

No Chemical Standards Required

Molecular Structure Directly Observed
Linear Concentration Response is Guaranteed
No Constant Calibration Necessary !!!

Superconducting Magnet
Superconducting special alloy coil Spectrometer
immersed in Liquid Helium at -269oC Radio-Frequency
and surrounded by a liquid 40-400 MHz
nitrogen dewar at 67K

Human, Yeast, and Bacterial Metabolomics

Cider/Beer/Wine =
Yeast-Bacteria Metabolites + Non-Assimilable Ingredients

Eg. "A primer to nutritional metabolomics by NMR spectroscopy

and chemometrics", F.Savorani,M. A. Rasmussen, M.S.
Mikkelsen, S. Balling-Engelsen, Food Research International,
54(1), 2013, Pages 1131-1145.
Ethanol-004-H
Ethanol in D2O nt=32
CH3-CH2-OH
CH3

Splittings = N+1 Rule

Adjacent to 2H = 3 Peaks

CH2
1 2 1

OH

Adjacent to 3H = 4 Peaks

1 3 3 1
0.90

1.96

3.00
6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
f1 (ppm)
 1 N=4
1
Succinic Acid
1

Ethylacetate A B C A N=3
B C N=3
N=2

W
Methanol N=3
W

L-Malic Acid D D N=2

E
E N=1

2 2
Isobutanol 3 4 N=6
2 4 N=2 3 N=1

5
8 N=2 9 N=3 5
Isoamylacetate 6 8 6 7 N=2
5 9 N=6
7 N=1

Citric Acid F F F N=4

Acetic Acid X N=3

Oligomers
Acetaldehyde Oligomers Y Z Y
Z
G N=6
2-3-butandiol H G
G H N=2
H

1-Propanol 11 12 N=2 11 N=2 10

12 N=3
10

9.8 9.6 5.2 5.0 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
 d, 5.32 ppm

Glucose d, 4.1 ppm

d, 5.21 ppm
Sucrose d, 5.4 ppm
Fructose Galacuronic Acid

Lactic Acid
Malic Acid d, 1.41 ppm Acetic Acid Succinic Acid Citric m, 2.94 ppm
m, 2.89 ppm s, 2.08 ppm s, 2.67 ppm

d, 0.88 ppm Ethanol

s, 3.32 ppm 1,3-propandiol

Glycerol Isobutanol Isoamyl alcohol t, 1.17 ppm p 1.76 ppm
Methanol m, 3.61 ppm d, 0.87 ppm
d 1.14 ppm
d, 1.56 ppm
s=singlet
d=doublet
t=triplet
p=pentet
m=multiplet
Alanine 2,3-butandiol
Examples of Major Protons Utilized Quinic Acid
for 1H Quantitative (qNMR) Analysis 2 x d 1.9 ppm
of Alcoholic Beverages
 Sigma-Aldrich Organic
TraceCERT® products are
characterized by:
•Manufactured according to
ISO/IEC 17025 and ISO Guide 34
•Certified Content by quantitative
NMR (qNMR)

•Superior level of accuracy,

calculated uncertainties and
lot-specific values
•Traceability to NIST
•Comprehensive
documentation delivered
with the product
(certification according to
ISO Guide 31)
 Water and Et-OH
1H NMR of Cider Product

Internal
Standard Ethanol

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
f1 (ppm)
 N=3
Beer-033-H
Goose Island Sofie - Farmhouse Saison HO-CH2-CH3
1H NMR in D2O 175ul + 100 ul MA
JHM-PNA-MVX300

Maleic Acid
Internal
Standard

N=2
100.00

367.75
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
f1 (ppm)
Calculations:
Standard Solution = 10mg Maleic Acid (MA) per 100ml of D2O Solution
Original Sample: The following volatile components were always obtained on a 175 ml beer sample as
the freeze drying process would compromise the amount present: ethanol, acetic acid, fusel alcohols
(isobutanol, isopentanol (isoamyl alcohol), and 1-propanol) ethyl acetate, methanol. The calculation
utilized to quantify these components was:

Component mg/L = 0.995 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp / 116.1) * (1,000,000 / 175)

Where, wt of maleic acid (MA) = 10mg, Icomp = Integration of component resonance, Ncomp = number of
protons integrated, IMA/NMA = 50 (MA integral set as 100), Mwcomp = molecular weight of component
molecule, MW of MA = 116.1 amu, the 1,000,000/175 factor rectifies the volumetric component of the
calculation to allow mg/L to be calculated. 0.995 represents the 99.5% purity of the maleic acid standard.
Freeze Dried Sample: The following non-volatile components were always obtained on a 1000 ml
freeze dried sample: lactic acid, succinic acid, malic acid, citrate, various amino acids, glucose, malt and
carbohydrates, glycerol. The calculation utilized to quantify these components was:

Component mg/L = 0.995 * 10 mg * ((Icomp / Ncomp) / 50) * (Mwcomp/ 116.1) * (1,000,000 / 1,000)

Where, wt of MA = 10 mg, Icomp = Integration of component resonance, Ncomp = number of protons

integrated, IMA/NMA = 50 (MA integral set at 100), Mwcomp = molecular weight of component molecule,
MW of MA = 116.1 amu, the 1,000,000/1,000 factor rectifies the volumetric component of the calculation
EthanolMix-002
Ethanol 45.0%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300

EthanolMix-003
Ethanol 22.5%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300

EthanolMix-004
Ethanol 11.25%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300

EthanolMix-005
Ethanol 5.625%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300

EthanolMix-006
Ethanol 2.812%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300

EthanolMix-070
Ethanol 0.1%ABV
1H qNMR 100ul MA
JCE-PNA-MVX300
x10

6.6 6.4 6.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
Example of Complex Mixture Quantitative Component Analysis – Bruker Winescreener

Information Copied from Bruker Website Materials

 Some Major Components of Cider
D-Fructose
How to ID and Quantify

D-Glucose

Sucrose

Glycerol

Ethanol

5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
 Apple Juice Analysis
Niagara Early Apple

Crab Apple

Fuji Apple Juice - 5 Days exposed

Gala Apple Juice

Granny Smith Apple Juice

Golden Delicious Apple Juice

Garnet Spy Apple Juice

Macoun Apple Juice

Crab Apple #2

Ring Sugar Protons

Sucrose, Glucose, Fructose
Sucrose Fructose
Glucose Malic

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
f1 (ppm)
 Concentration (g/L)
Apple Variety Sucrose Glucose Xylose Fructose Red Sugar Non-Red Sugar Total Sugar Malic Acid
1 Cavelle Blanc 125.9 14.1 0.8 120.2 135.1 125.9 261.0 20.7
2 Fall Pippin 63.5 8.1 0.7 115.0 123.9 63.5 187.4 9.5
3 Dr Mathews 33.6 6.6 0.4 86.2 93.2 33.6 126.8 6.3
4 Chieftan 59.2 7.7 0.6 85.0 93.3 59.2 152.6 8.4
5 Pink Pearl 56.6 7.7 0.6 84.4 92.6 56.6 149.2 14.8
6 Oakin Pin 52.2 17.1 0.4 111.9 129.4 52.2 181.6 25.7
7 King David 48.3 7.7 0.7 100.4 108.8 48.3 157.1 10.8
8 Bennoni 51.7 8.5 1.2 97.7 107.4 51.7 159.2 9.6
9 Green Newtown Pippin 86.6 9.8 0.3 86.7 96.8 86.6 183.4 19.8
10 Ashmaead Kernel 78.8 11.4 0.5 109.3 121.2 78.8 200.0 14.5
11 English Golden Harvey 104.5 11.2 0.4 127.2 138.8 104.5 243.3 22.2
12 Yates 34.9 9.6 0.3 74.2 84.2 34.9 119.0 7.5
13 Spitzberg 61.5 10.5 0.8 123.0 134.3 61.5 195.8 9.5
14 Tompkin's King 59.1 13.3 0.6 137.9 151.7 59.1 210.8 14.9
15 Golden Russet 68.7 17.5 0.7 170.6 188.8 68.7 257.6 13.3
16 Decio 78.5 7.1 0.4 69.9 77.4 78.5 155.9 7.8
17 Edwards VII 49.2 10.1 0.6 137.8 148.5 49.2 197.7 10.5
18 Rhode Island Greening 48.1 9.9 0.3 88.6 98.9 48.1 147.0 9.0
19 Baldwin 105.8 6.1 0.3 81.6 87.9 105.8 193.8 17.6
20 Starr 8.4 6.3 0.4 74.1 80.8 8.4 89.2 9.2
21 Razor Russet 67.3 11.0 0.6 125.7 137.2 67.3 204.5 9.8
22 Gold Rush 62.8 4.9 0.3 67.6 72.8 62.8 135.6 11.0
23 Jewitt Nodhead 45.5 12.1 0.7 131.5 144.4 45.5 190.0 8.9
24 American Beauty Sterling 76.6 8.0 0.5 86.1 94.5 76.6 171.1 13.4
25 Kidds Orange Red 42.5 8.7 0.7 119.2 128.6 42.5 171.1 8.6
26 Opalescent 42.7 9.0 0.7 94.8 104.6 42.7 147.3 8.0
27 Ben Davis 30.4 6.2 0.5 74.9 81.5 30.4 111.9 6.4
28 Chestnut Crab 7.4 7.9 0.6 94.1 102.6 7.4 110.0 9.7
29 Scarlet Crofton 62.9 9.3 0.9 105.9 116.1 62.9 179.0 9.8
30 Quinte 39.3 6.4 0.8 70.9 78.1 39.3 117.4 9.6
31 Barnack 69.8 14.2 0.8 118.2 133.1 69.8 202.9 10.0
32 Sweet Winter Pennock 31.8 7.4 0.3 86.6 94.3 31.8 126.1 6.1
33 Bancroft 56.3 5.3 0.7 116.3 122.4 56.3 178.7 18.7
34 Titus Pippen 33.7 7.2 0.8 101.6 109.6 33.7 143.3 9.5
35 Spy 94.4 14.5 1.0 146.3 161.8 94.4 256.2 14.1
36 Pearl Apple 91.8 8.6 0.7 133.1 142.4 91.8 234.3 20.0
37 Golden Noble 67.1 12.4 1.2 124.6 138.1 67.1 205.3 24.1
38 Devonshire Quarendon 23.0 10.4 0.8 105.8 117.0 23.0 140.0 9.1
39 Virginia Beauty 66.5 17.6 0.8 113.4 131.8 66.5 198.3 13.2
40 Winter Banana 24.1 17.1 0.6 108.7 126.4 24.1 150.5 8.9
41 Marie Joseph 20.0 3.4 0.2 38.5 42.1 20.0 62.1 6.9
5.4 5.3 5.2 5.1 5.0 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 42 3.3
Mother 3.2 3.1 3.0
70.8 2.9
9.2 0.92.8 119.3 2.7
129.3 70.8 200.1 10.8
f1 (ppm) 43 Pitmaston Pineapple 50.6 26.3 1.1 196.2 223.6 50.6 274.2 16.2
44 Roxburg Russet 78.8 16.6 0.7 141.4 158.7 78.8 237.6 14.1
45 Shiawasse Beauty 34.8 5.7 0.6 129.0 135.3 34.8 170.1 13.6
46 Wagener 77.1 9.8 0.5 111.6 121.8 77.1 198.9 13.0

54 Apple Juices of Heirloom Variety Apples 47

48
49
Sutton Beauty
Yellow Newtown Pippin
Colamer
47.5
74.2
29.8
9.3
17.0
5.0
0.4
1.1
0.6
102.5
138.8
97.1
112.2
157.0
102.6
47.5
74.2
29.8
159.7
231.1
132.5
10.0
10.4
11.1

Apples Obtained at Cider Days – Franklin MA

51 Montreal Peach 72.9 10.2 0.6 144.5 155.3 72.9 228.2 9.3
52 Goof 17.7 15.8 0.6 103.7 120.1 17.7 137.8 8.8
53 Ellisons Orange 89.9 9.3 0.7 95.3 105.2 89.9 195.1 10.7
54 Granny Smiths 53.7 9.2 0.1 64.1 73.3 53.7 127.0 18.7
55 Gala 45.7 23.9 0.7 191.2 215.8 45.7 261.5 7.2
56 Fuji 8.3 20.2 0.4 112.2 132.8 8.3 141.1 2.9
Analysis of Other Fruit Juices
Grape

Blueberry

Blackberry

Apricot

Strawberry

Raspberry

Navel Orange

Mirabelle Plum

Lime

Lemon

Cranberry

Cherry

Grapefruit

Quince

Pomegranate

Pineapple

Orange

Plum

Mango

Mandarin

Apple

5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0
f1 (ppm)
 Some Major Components of Cider
D-Fructose
How to ID and Quantify

D-Glucose

Sucrose

Glycerol

Ethanol

5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
 N=0.53 N=2.02 Total Labile Proton Integral = 7

D-Fructose Single doublet or series of peaks utilized to quantify fructose

D-Glucose

anomeric protons
utilized to quantify
glucose in cider
a b

Sucrose Which fructose peaks are utilized to calculate

the concentration depends on the presence or
Anomeric proton non-presence of sucrose as it has a signal that
utilized to quantify overlaps with this region.
sucrose

5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0
f1 (ppm)
 Red - Fructose
Blue - Glucose
Green - Sucrose

Sucrose

Glucose Fructose

5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1
f1 (ppm)
 Nutrient Effect on Cider Chemistry During Fermentation
Grote-009-H_FD
Cider #3 Fructose
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300
Glucose

Sucrose Malic
Acid

Grote-008-H_FD
Cider #2
Parallel Fermentation of Three Ciders from Sample Juice
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-007-H_FD
Cider #1 Cider#1 – Nutrient Depleted
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300 Cider #2 – Control
Cider #3 – Nutrient Added

6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
f1 (ppm)
Grote-009-H_FD
Cider #3
YAN Observed in Cider #3
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300
After addition of nutrients
Valine
Alanine Luecine
Glutamic
Isoleucine
Acid

Grote-008-H_FD
Cider #2
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-007-H_FD
Cider #1
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.04.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
f1 (ppm)
Grote-022-H Aromatic Proton Signals from Pure Amino Acids and O-Ferm Nutrient Product
Scott Labs O-Ferm
1H NMR in D2O 100ul MA
JCE-PNA-MercPlus300

HO
13
AminoAcid-L-Tyrosine 12 11
L-Tyrosine in acidified D2O 11,10 8,9
JCE-PNA-MVX300
10 9 O
7
8 5 4 OH
6
3 2

NH2
OH 1
6
O
7
4 9
AminoAcid-L-Tryptophan 9
NH
L-Tryptophan in Acidified D2O 12 2
NH2
JCE-PNA-MVX300 10
5
3 1
15 13 11 14
13 8

15 11
14

12 10
10,11
AminoAcid-L-Phenylalanine 11 8 O
L-Phenylalanine in acidified D2O 6 8,9
JCE-PNA-MVX300 9 7 3 OH
5
4 2 12
NH2
1

10
NH
11
AminoAcid-L-Histidine 9
O
L-Histidine in acidified D2O N 5
JCE-PNA-MVX300 10 8 7 9
3
OH
4 6
2

NH2
1

9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7
f1 (ppm)
Aliphatic Peak Region for Amino Acids Compared to Cider #3 and Nutrient
Ala IsoLeu, Leu
Val, IsiLeu

Grote-022-H Glu Thr

Scott Labs O-Ferm
Glu Leu

O CH3
1 8

AminoAcid-L-Valine 2 6 7 8
6 HO 3 CH3
4 7

H2 N
5 OH
3
1
AminoAcid-L-Threonine H3 C 2
1
4 NH2
5

O 6
O 7
1 OH
AminoAcid-L-Leucine
8 9
2 6
CH3 7,6 8
HO 4 7 9
3
O H3 C
CH3 1 8
H2 N 8
5 2 5 CH3
3 7 9 9 8
AminoAcid-L-IsoLeucine HO
4

5 7 7 NH2
6
O
7
4 OH
6
AminoAcid-L-Glutamic Acid H2 N 2

5 3 1
3 5

8 O
9 O
HO 1
10
AminoAcid-L-Alanine 6 2 CH3
6
HO 4
3

NH2
5

Grote-009-H_FD Val, IsoLeu

Glu Ala Thr Leu
Cider #3 Glu
Leu

2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7
f1 (ppm)
Grote-019-H_FD
Cider #1 1-5-18
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300 Gly
Mal Succinate

Grote-016-H_FD
Cider #1 Pre-Rack 11-30-17
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300

Grote-013-H_FD
Cider #1 11-22-17
1H NMR in D2O 1000ul FD+100ulMA
JCE-PNA-MVX300

Grote-010-H_FD
Cider #1 11-10-17 - 1 Week Ferment
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-007-H_FD
Cider #1
1H NMR in D2O 1000ul FD + 100ul MA
Fru
JCE-PNA-MercPlus300
Suc Glu

5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4
f1 (ppm)

Cider #1
NMR Data over Fermentation
Grote-020-H_FD
Cider #2 1-5-18
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300 Gly
Mal Succinate

Grote-017-H_FD
Cider #2 Pre-Rack 11-30-17
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300

Grote-014-H_FD
Cider #2 11-22-17
1H NMR in D2O 1000ul FD+100ulMA
JCE-PNA-MVX300

Grote-011-H_FD
Cider #2 11-10-17 - 1 Week Ferment
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Grote-008-H_FD
Cider #2
1H NMR in D2O 1000ul FD + 100ul MA
Fru
JCE-PNA-MercPlus300
Suc Glu

5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4
f1 (ppm)

Cider #2
NMR Data over Fermentation
Grote-021-H_FD
Cider #3 1-5-18
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300
Gly Mal Succinate

Grote-018-H_FD
Cider #3 Pre-Rack 11-30-17
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MVX300

Grote-015-H_FD
Cider #3 11-22-17
1H NMR in D2O 1000ul FD+100ulMA
JCE-PNA-MVX300

Grote-012-H_FD
Cider #3 11-10-17 - 1 Week Ferment
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300
Fru

Grote-009-H_FD
Cider #3
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

Suc Glu

5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4
f1 (ppm)

Cider #3
NMR Data over Fermentation
Comparison of Sugar Data for 3 Ciders over Course of Fermentation
 L
Ac

Different Dry Cider Styles

Spanish and Wild Fermentations

1,3-PD
F
Succ
EA
EA M

Millstone-001-H
Millstone Thistle Mead 8.5% ABV
1H NMR in D2O 175ul+100ul MA
JCE-PNA-MVX300

4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8
f1 (ppm)
 Ac Lac
Cider-029-H
Hudson Valley Farmhouse Cider Bourbon Barell Aged 6.8% ABV
NY Farmhouse Barrel Aged
1H NMR in D2O - 175ul Cider - 100ul MA
Succinate
JHM-PNA-MVX300 Methanol

Fusel

Cider-28-H
Cidre Breton Traditionnel 4.5% ABV
Breton Cidre
1H NMR in D2O - 175ul Cider - 100ul MA
JHM-PNA-MVX300

Mal

Cider-27-H
Pitchfork Hard Cider 6.9% ABV
NY Dry Cider
1H NMR in D2O - 175ul Cider - 100ul MA
JHM-PNA-MVX300 Gly

Cider-30-H
Angry Orchard Hard Cider Summer Honey 5% ABV
Back Sweetened
1H NMR in D2O - 175uL Cider 100uL MA
JHM-PNA-MVX300

Examples: ciders with different chemical characteristics – sugar concentration + malolactic

5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
Cider-069-H
Propionic Acid
Sider Avalon 5.5%ABV
Spanish Sidra 2
1H NMR in D2O 175ul + 100ul MA
JCE-PNA-MVX300

Cider-065-H
Glycerol
Sidreria Gurutzeta Sagardoa Sidra Natural 6%ABV
Spanish Sidra 1
Basque Cider, Spain 1H NMR in D2O 175ul +100ul MA
Absent
JCE-PNA-MVX300
Hydroxypropionic Acid

Cider-064-H
Millstone Cellars Sidra Americana Batch 002 7%ABV
“American Sidra”
Monkton MD pH 3.63 1H NMR in D2O 175ul +100ul MA
JCE-PNA-MVX300

Ethyl lactate
Ethyl acetate 1,3-propandiol 1-propanol
Additives-002-H
1,3-Propanediol Lot DT17F00009-9390
1H NMR in D2O
JCE-PNA-MercPlus300

Unique Character of Sidra and Other Wild Fermented Cider – metabolism of glycerol utilization
6.0 5.8 5.6 5.4 5.2 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
 Anaerobic Fermentation of Glycerol
Seems to occur in all Sidra and in many wild fermentations
Ethanol Fermentation Process that ferment sugars “to dryness”.
Maple Syrup Grade A Ac Chemistry observed in
Honey and Maple Syrup
Succinic
Malic

Lac
Et
Suc

3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
Glu f1 (ppm)

Wildflower Honey

raf+imt+im3
+mlb+plt
trn
kjb+mlz
trn+mlt

Fru 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3
f1 (ppm)
Glu

Honey = Terroir Fingerprint in Residual Sugar Distribution

kjb, kojibiose; mlz, melezitose; trn, turanose; mlt, maltulose; raf, rafinose; imt, isomaltose;
Non-Fermentable Sugars plt, palatinose; im3, isomaltotriose; mlb, melibiose; glu, glucose

5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4
f1 (ppm)
Cyzer

Honey

Cyzer
Residual Sugars

Honey
Sugar Distribution

5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9

f1 (ppm)

6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6
f1 (ppm)
 Cyzer and Mead - High Concentrations of Unused Nutrient Amino Acids
AminoAcid-L-Tyrosine
L-Tyrosine in acidified D2O
JCE-PNA-MVX300

AminoAcid-L-Tryptophan
L-Tryptophan in Acidified D2O
JCE-PNA-MVX300

AminoAcid-L-Phenylalanine
L-Phenylalanine in acidified D2O
JCE-PNA-MVX300

AminoAcid-L-Histidine
L-Histidine in acidified D2O
JCE-PNA-MVX300

Cyzer-001-H_FD
Cyzer 14%ABV
1H NMR in D2O 1000ul FD + 100ul MA
JCE-PNA-MercPlus300

8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6
f1 (ppm)
 Mystery Solved?
Batch 2 – supposedly the same cider.
Lactic
Cider-042-H Contains twice as much sorbate.
DownEast Original Blend Cider Batch Y Was Batch 1 under-additized causing continued
Batch 2 – “good”
1H NMR in D2O 175ul Cider + 100ul MA
Fermentation and explosions?
JCE-PNA-MVX300

New Mystery – why has malolactic fermentation

taken off – juice handling
More Sorbate
Acetic

Cider-041-H_RepeatRun Malolactic
DownEast Original Blend Cider Batch X Good Can
Batch 1 – “good”
1H NMR in D2O 175ul Cider + 100ul MA
JCE-PNA-MVX300

Cider-041-H_Bad_RepeatRun Mystery: Obvious reduction of sugars and

DownEast Original Blend Cider Batch X Overpressurized Can
Batch 1 – Exploding Can
1H NMR in D2O 175ul Cider + 100ul MA
production of more ethanol observed and
JCE-PNA-MVX300 compared to “good” can – didn’t explain exploding can

Production and Quality Issues

6.0 5.8 5.6 5.4 5.2 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8
f1 (ppm)
Production and Quality Issues
Examples of Sugar Analysis of Commercial Ciders – Part of Dryness Scale?
Organic Acids Distribution