Food Chemistry 188 (2015) 149–160

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Glucose: Detection and analysis q

A.L. Galant, R.C. Kaufman, J.D. Wilson ⇑
USDA-ARS, Grain Marketing and Production Research Center, Manhattan, KS 66502, United States

a r t i c l e i n f o a b s t r a c t

Article history: Glucose is an aldosic monosaccharide that is centrally entrenched in the processes of photosynthesis and
Received 16 September 2014 respiration, serving as an energy reserve and metabolic fuel in most organisms. As both a monomer and
Received in revised form 10 April 2015 as part of more complex structures such as polysaccharides and glucosides, glucose also plays a major
Accepted 17 April 2015
role in modern food products, particularly where flavor and or structure are concerned. Over the years,
Available online 23 April 2015
many diverse methods for detecting and quantifying glucose have been developed; this review presents
an overview of the most widely employed and historically significant, including copper iodometry, HPLC,
Keywords:
GC, CZE, and enzyme based systems such as glucose meters. The relative strengths and limitations of each
Glucose
Glucose meters
method are evaluated, and examples of their recent application in the realm of food chemistry are
Iodometry discussed.
Detection Published by Elsevier Ltd.
Quantification

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
2. The discovery of glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3. Copper-iodometric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4. Enzymatic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.1. Glucose oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.2. Hexokinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.3. Glucose dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.3.1. Glucose-6-phosphate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.4. Glucose 1-dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.5. Quinoprotein glucose dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.6. FAD-glucose dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5. Glucose meters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
6. Non-enzymatic glucose sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
7. HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
8. Capillary zone electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
9. GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
10. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
11. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Abbreviations: BGE, background electrolyte; C(Z)E, capillary (zone) electrophoresis; DCPIP, 2,6-dichlorophenol indophenol; ELSD, evaporative light scattering detection;
FAD, flavin adenine dinucleotide; FID, flame ionization detector; GC, gas chromatography; GDH, glucose-1-dehydrogenase; G6P, glucose 6-phosphate; G6PDH, glucose 6-
phosphate dehydrogenase; GOPOD, glucose oxidase peroxidase; GOx, glucose oxidase; HILIC, hydrophilic interaction chromatography; HPLC, high performance liquid
chromatography; MS, mass spectrometry (mass spectrometer); NPD, nitrogen–phosphorus detector; NEGS, non-enzymatic glucose sensor; PAD, pulsed amperometric
detection; PQQ-GDH, quinoprotein glucose dehydrogenase; RID, refractive index detector; RP, reversed phase; SEC, size exclusion chromatography; TMS, trimethylsilyl.
q
U.S. Department of Agriculture, Agricultural Research Service, Plains Area, is an equal opportunity/affirmative action employee and all agency services are available
without discrimination. Mention of firm names or trade product does not constitute endorsement by the U.S. Department of Agriculture over others not mentioned.
⇑ Corresponding author.
E-mail address: jeff.d.wilson@ars.usda.gov (J.D. Wilson).

http://dx.doi.org/10.1016/j.foodchem.2015.04.071
0308-8146/Published by Elsevier Ltd.
150 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
1. Introduction
Among the many biological compounds found in nature, glu-
cose is arguably one of the most critical for life. As the primary fuel
for glycolysis and the downstream pathways of aerobic and anaer-
obic respiration, glucose is responsible for generating much of the
energy potential required for successful growth and reproduction.
In plants and cyanobacteria, glucose is produced from water and
carbon dioxide via photosynthesis and condensed to form starch.
The starch may be stored as an energy reserve, or broken down
and used as a substrate for the synthesis of a wide variety of sac-
charides – including sucrose and cellulose. In foods, particularly
those that are plant-derived, these diverse carbohydrates con-
tribute greatly to texture and flavor, while serving as a secondary
source of energy post-consumption.
There are a myriad of analytical assays in which glucose can be
determined in food products and food product precursors. The
large number of assays presents a challenge: how might glucose
be unambiguously identified and quantified, given the nearby
presence of so many biochemically similar molecules? In the
mid-18th century, rudimentary colorimetric assays which could
nonspecifically detect reducing sugars in blood and urine were first
conceived. As technology and knowledge of carbohydrate chem-
istry advanced, new methods which were specific for select combi-
nations of monosaccharides or for glucose alone were established.
Although developed with an eye toward the diagnosis and moni-
toring of human diabetes, these methods were equally effective
for quantifying glucose in plant extracts and food products such
juices and honey.
Today, a variety of unambiguous methods for detecting and
quantifying glucose in assorted food matrices exist. These methods
may be broadly grouped into two main categories: enzymatic
approaches – encompassing both spectrophotometric assays and
glucose meters – and non-enzymatic instrumentation such as
HPLC systems and their associated detectors. While the former
group is glucose specific, the latter is broadly adaptable and may
be used to detect not only glucose, but also an assortment of other
carbohydrates. In this review we provide an overview of enzymatic
glucose detection systems, and discuss their relative strengths and
weaknesses. We also examine the assorted chromatographic sys-
tems and electrophoretic-based systems (HPLC, GC, CZE) and asso-
ciated detectors which may be used for glucose detection and
analysis. A synopsis of the events leading up to the discovery of
glucose and an overview of the semi-historical wet chemistry
methods associated with its early quantification are also provided.
A limit of detection table with references has been included to
allow readers more rapid access (Table 1).
Table 1
Glucose limits of detection with references.
Method Limit of detection
Glucose oxidase 0.36–23.4 g/L
Glucose oxidase 0.18–3.6 g/L
Non-enzymatic glucose sensors 0.54 mg/L
0.94 mg/L
0.018–180 mg/L
HPLC–RID 100–5000 mg/L with LOD of 67 mg/L
0.1–20 g/L range with LOD of 0.16 g/L
29 lg/mL
HPLC–PAD 0.036 mg/L
HPLC–ELSD 0.37 lg/L
HPLC–fluorescence and UV 9 lg/L
RP–HPLC 0.023 lg/L
Capillary zone electrophoresis
2. The discovery of glucose
The use of carbohydrates (‘‘sugars’’) as a flavor enhancer and/or
fermentation substrate is a practice steeped in history (Fig. 1). The
sugarcanes (Saccharum L.) and the sorghums (Sorghum L.), both
genera comprised of tall, perennial grasses with sugar-rich stems.
Modern sorghums are thought to originate from the Sahel region
of Africa, whereas sugar cane is indigenous to Southeast Asia, in
particular New Guinea. Sugar cane was first domesticated between
6000 and 8000 years ago; the stalks at this time were chewed so as
to extract the sugar. By 800 B.C., sugarcane cultivation had spread
across India, Mesopotamia, China, and the Pacific Islands; by 800
A.D., crude methods for extracting and processing cane sugar were
in place. Sugar was first introduced to Europeans by crusaders
returning from the Holy Lands prior to 1200 A.D., and soon traded
at prices reserved for other rare spices such as saffron and pepper.
That demand for sugar far outpaced supply, combined with the fact
that sugarcane could be grown only in the southernmost reaches of
Europe, led to the crop factoring heavily into the European con-
quest and ensuing cultivation of the New World.
As foreign colonization leveled off, and foreign holdings began
to seek independence, alternative, cost-effective sources of sugar
for the European markets were sought. In 1600, the French agrono-
mist Olivier de Serres noted that ‘‘The beet on being cooked yields
a syrup which is beautiful to look at on account of its vermillion
color’’ (Palmer, 1913). This observation was expanded upon by
the German chemist Andreas Marggraf, who succeeded in crystal-
lizing sugar from beet roots in 1717 and then subsequently from
raisins. Although Marggraf does not provide details of his raisin
experiment, some later reports list it as the first successful
extraction of glucose (Fontenelle and Mairan, 1741). In 1838, the
French chemist Jean Baptiste Andre Dumas coined the term
‘‘glucose’’ as a name for the sugar that could be extracted from
grapes, raisins, and other fruit. Although at the time it remained
unclear whether this ‘‘glucose’’ was a single compound or a mix-
ture, Dumas predicted:
‘‘I believe that glucose will be found identical, whatever be its
origin; whether from honey and fruits, or from diabetic urine,
or produced by chemical action upon starch and ligneous fiber.’’
[Doc. No. 146; Chemical Analysis of Sugar]
While it was evident by the mid-18th century that ‘‘glucose’’
extracted from different sources rotated light differently and pos-
sessed differing degrees of sweetness, it was not until Emil
Fischer deduced the structures of all of the major simple sugars
in 1891–94 that glucose was conclusively identified.
An overview of the history of glucose would not be complete
without mention of its role in human diabetes. The earliest
References
‘‘BRENDA; The Comprehensive Enzyme
Information System’’ (2014)
Yuen and McNeil (2000)
Hall and Keuler (2009)
Wei et al. (2014)
Toghill and Compton (2010)
Carballo et al. (2014)
Eyéghé-Bickong et al. (2012)
Ma et al. (2014)
Gangola et al. (2014)
Ma et al. (2014) and Shanmugavelan et al. (2013)
Shaw and Wilson (1983)
Dai et al. (2010)
 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
Domescaon of sugarcane
6000-4000 B.C
Methods for processing
sugarcane are developed
800 B.C – 800 A.D.
• 230 B.C.
Term ‘diabetes’ coined
• 100 B.C.
Sugarcane introduced to China
• 500 A.D.
Type I and type II diabetes differenated
• 700 A.D.
Sugarcane introduced to Europe
Fig. 1. Timeline of select major events in the history of glucose.
possible reference to the disease comes from a 2500 year old
Egyptian papyrus excavated from Thebes in 1862 and translated
by George Ebers. A contemporary report by Indian physicians
described a condition in which ants and flies are attracted to
human ‘‘honey urine.’’ This condition was first termed ‘diabetes’
by Appolonius of Memphis c. 230 B.C. and by 500 A.D. two
Indian physicians – Susruta and Charaka – had differentiated
between the type 1 and type 2 forms of the disease (Zajac,
Shrestha, Patel, & Poretsky, 2010). In the same year that Dumas
gave glucose its name, the physician George Rees isolated sugar
in excess from the blood of a patient with diabetes (Rees, 1838).
Methods for quantifying this sugar in blood and urine soon fol-
lowed; most relied on the ability of glucose to reduce copper, yield-
ing a colorimetric or titratable end-product. These early wet-
chemistry methods, laid the groundwork for the development of
the non-enzymatic glucose sensor, an electrode of variable compo-
sition which shows great promise for both diabetes- and food-
based glucose testing.
3. Copper-iodometric methods
Early methods for the quantification of glucose relied on its
ability to act as a reductant in solution. Although an assortment
of metal ions could oxidize the glucose carbonyl group, copper
(Cu2+) was the most popular due to the potential for formation of
stable precipitates or colorimetric end-products. Benedict’s solu-
tion was one of the first widely-used reagents to take advantage
of the relevant chemistry, employing sodium carbonate in the
presence of copper citrate or tartrate to precipitate Cu2O
(Benedict, 1908). This chemistry was co-opted by Otto Folin and
Hsien Wu, who coupled the reduction of copper to the oxidation
of phosphomolybdic acid (the Folin–Wu method). The result was
a blue end-product, which was stable for several days, and which
could be compared directly against solutions of known starting
sugar content (Folin & Wu, 1919). One short-coming of
Benedict’s solution and the Folin–Wu method was the inability
to differentiate glucose from other reducing sugars in a complex
solution. To address this issue, Cajori devised a method by which
excess iodine was first reduced by glucose, and subsequently
titrated against thiosulfate to determine the quantity of glucose
present. Any fructose and sucrose present would not react with
iodine, but fructose that remained after the glucose had been con-
verted to gluconic acid could be reacted with copper as previously
described by Folin and Wu. Next, any sucrose present could be
quantified by selective hydrolysis and subsequent oxidation of lib-
erated glucose by iodine. Finally, any maltose present was quanti-
fied by comparing the glucose reducing power of the solution both
before and after introduction of a yeast maltase (Cajori, 1922).
Further optimization of the iodometric method was carried out
by Shaffer, Somogyi, and Nelson. In particular, the procedure was
151
Structure of glucose determined
• 1894
Sugar crystallized from beets
• 1717
• 1516
First sugar mill established on Hispaniola
• 1838
Term ‘glucose’ coined
• 1908
Benedict’s soluon developed
• 1957
First commercial
glucometer marketed
adapted to quantify smaller quantities (10 lg) of glucose, and a
colorimetric arsenomolybdate reagent that was used in conjunc-
tion with early spectrophotometers was developed (Shaffer &
Somogyi, 1933).
Although the earliest methods for detecting glucose were devel-
oped with diabetic blood and urine in mind, early plant bio-
chemists and food technologists rapidly adopted the techniques
for their own ends. Reports on the syrup composition of corn stalks
and the sugars in nonspecific plant extracts utilized a repurposed
colorimetric picric acid method developed by Benedict
(Thomas & Dutcher, 1924; Willaman, Burr, & Davison, 1924).
Investigations into the sugar and starch components of white pota-
toes combined paper chromatography with a micro-adaptation of
the iodometric glucose method, while total reducing sugars and
sucrose were determined in apples using reduced copper titrated
against potassium dichromate (Schwimmer, Bevenue, Weston, &
Potter, 1954). In more recent times, use of these wet chemistry
techniques has been limited, due to the widespread availability
of analytical instrumentation which simplifies the process of sepa-
rating and quantifying the reducing and non-reducing sugars. But
the basic redox chemistry associated with reducing sugars, and
in particular the use of copper as an oxidant, has seen resurgence
in the form of non-enzymatic glucose sensors (see below).
4. Enzymatic methods
In solution, glucose may exist as one of two possible anomers –
termed a-, and b-, or as the open-chain glucose aldehyde (Fig. 2).
When allowed to reach equilibrium at pH 7 and 25 °C, approximately
63% of the glucose will adopt the b-glucopyranose conformation,
with 37% existing as the a-glucopyranose, and less than 1% existing
as either the aldehyde or as glucofuranose. Interconversion between
the anomers may occur freely (albeit slowly) or via the catalytic
activity of glucose mutarotases. Most enzymes preferentially bind
either the a or b form, but a small subset are able to utilize either
anomer as the situation demands. Because glucose serves as the
lynchpin of a number of downstream metabolic pathways – includ-
ing glycolysis/gluconeogenesis, the pentose phosphate pathway,
production of other carbohydrates such as cellulose and sucrose,
etc. – there exist a number of enzymes which accept it as a substrate
and produce a detectable/quantifiable product and/or by-product. In
particular, the activities of glucose oxidase and hexokinase/glucose
have been co-opted in the development of spectrophotometric and
colorimetric glucose assays.
4.1. Glucose oxidase
Glucose oxidase (GOx; EC 1.1.3.4) is a dimeric enzyme which
catalyzes the conversion of b-D-glucose to D-glucono-1,5-lactone
as part of the pentose phosphate pathway.
152 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
Fig. 2. Anomers and aldehyde form of D-glucose.
GOxðFADH2 Þ þ O2 ! GOxðFADÞ þ H2 O2
b-d-glucose þ GOxðFADÞ ! d-glucono-1; 5-lactone þ GOxðFADH2 Þ
Each subunit of the enzyme contains one iron atom and one fla-
vin adenosine dinucleotide cofactor, both of which are required for
catalytic activity. The active site is specific for the b anomer of glu-
cose; however non-specific activity in the presence other sugars,
including mannose and galactose, has been reported. Although
activity is possible at a range of pHs, GOx generally prefers acidic
conditions. The commonly-used GOx from Aspergillus niger for
instance, has a preferred pH range of 4–7 with an optimum around
5.5. Unlike many inorganic detection systems, GOx is active at a
wide range of physiologically relevant glucose concentrations; Km
values between 2 mM and 130 mM have been reported for b-glu-
cose, implying a much wider range for potentially detectable activ-
ity (‘‘BRENDA; The Comprehensive Enzyme Information System’’,
2014).
Because one of the end products is H2O2, GOx reactions can be
coupled to a wide variety of detection systems for monitoring glu-
cose concentration. Popular methods involve the use of a peroxi-
dase, such as horse-radish peroxidase, to drive the oxidation of a
chromophore. The Trinder method, an older means of quantifying
glucose in serum and blood, utilized peroxidase in conjunction
with phenol/4-aminoantipyrine to generate a red chromophore
with strong absorbance between at 505–520 nm (Trinder, 1969).
Commercial kits now containing a wide variety of fluorescent
and visible light chromophores, including amplex red (10-acetyl-
3,7-dihydroxyphenoxazine, Em: 585 nm;), phenol/4-aminoan-
tipyrine (A: 514 nm;), and o-dianisidine/sulfuric acid (A:
540 nm;). Collectively, these commercial coupled systems are
often referred to as GOPOD (Glucose Oxidase PerOxiDase) reac-
tions. The sensitivity and specificity of these kits depends upon
the purity of the enzyme preparations, but accuracy over a
dynamic range of 1–20 mM glucose is generally quite good (Yuen
& McNeil, 2000). With extreme care, detection may be possible
down to 3 lM (Hall & Keuler, 2009). Because of the requirement
ð1Þ for oxygen in the GOx reaction, glucose sensing using this method-
ology is not under anaerobic conditions such as those associated
with fermentation. Otherwise, the general methodology is suffi-
ciently adaptable and scalable such that it has found widespread
ð2Þ
use throughout the field of food chemistry. Recent analyses which
incorporate glucose oxidase based glucose detection include a
quantification of the resistant and total starch in 22 different food
staples including bananas and yams (Moongngarm, 2013), and a
new assay for quantifying b-glucan content (Danielson et al.,
2010). Of interest is also a means by which glucose can be detected
in food products via rheological changes brought on by the GOx-
mediated formation of calcium-alginate gels (Liu, Javvaji,
Raghavan, Bentley, & Payne, 2012).
With regard to the GOx reaction, recent improvements in the
field of nanochemistry have led to the development of an alternate
glucose-sensing fluorophore: carbon nanodots. Like peroxidase,
these dots – which may be synthesized using an autoclave from
a variety of sources – react with the H2O2 produced by the GOx
reaction. The result is a quenching of the nanodot photolumines-
cence or direct oxidation of an introduced fluorophore (e.g.
3,30 ,5,50 -tetramethylbenzidine, Em: 652 nm) that is linear with
respect to gluconolactone evolution, and sensitive down to
5.2 lM glucose (Wei et al., 2014). The primary advantage of carbon
nanodots over other fluorophores is their stability: while nanodots
are stable at pHs between 0 and 12 and temperatures between 0 °C
and 90 °C, horse-radish peroxidase activity is reduced by approxi-
mately 80% relative to nanodots by temperatures above 60 °C, and
by 40–75% below pH 5 or above pH 10 (Shi et al., 2011). Additional
work is still needed to determine how nanodots will hold up to
conditions in a blood or food-based matrix, but the methodology
shows promise to date.
4.2. Hexokinase
Rarely used as an independent means of quantifying glucose,
hexokinase (EC 2.7.1.1) is typically employed as the first enzyme
in a two-step detection method that is coupled to glucose 6-phos-
phate dehydrogenase (G6PDH).
 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160 153

d-glucose þ ðNÞTP ! Glucose-6-phoshate þ ðNÞDP ð3Þ to addition of hexokinase/G6PDH. Failure to ensure this may result
in an overestimation of starting glucose concentration. Like the
Hexokinase catalyzes the (N)TP-dependent phosphorylation of
GOPOD reactions, hexokinase/G6PDH can be purchased as part of
glucose at the 6-carbon position. The enzyme exhibits low speci-
a commercial kit (Megazyme; A: 340 nm). The main advantage of
ficity, and will readily accept mannose, and many other carbohy-
these methods is twofold: first, the cost barrier for purchasing a
drates in addition to glucose (‘‘BRENDA; The Comprehensive
spectrophotometer is typically lower than that for an HPLC or GC.
Enzyme Information System’’, 2014). Several different triphosphate
Second, spectrophotometric methods are more tolerant of the pres-
cofactors, including UTP and GTP are accepted, with a penalty to
ence of non-absorbing soluble proteins, carbohydrates, etc. that
enzyme turnover rate. A subset of hexokinases, termed glucoki-
may plug a column or fail to fully volatilize. As hexokinase has a
nases (EC 2.7.1.2), are specific only for glucose, but require a
km value for glucose between 10- and 1000-fold lower than the cor-
100-fold higher substrate concentration for comparable activity.
responding glucose oxidase (e.g. A. niger hexokinase km: 0.023 mM;
Provided that the second enzyme employed is specific for glu-
A. niger glucose oxidase km: 26 mM), hexokinase coupled systems
cose-6-phosphate (G6P) and that ADP formation is not used as a
may be more appropriate in situations where low glucose concen-
measure of glucose-specific activity (such as via a coupled pyru-
trations are expected. Ultimately, the choice of an appropriate
vate kinase/lactic dehydrogenase system), then the lack of speci-
detection system may hinge upon knowledge of the background
ficity will not present an issue. Both glucokinases and
absorption of the solution of interest, and selection of a spectropho-
hexokinases are maximally active between pHs 7 and 9, and mod-
tometric glucose detection system that avoids that problematic
erately active (30–70%) at pH 6. Either alpha or beta-glucopyranose
wavelength range. In a recent series of experiments involving deter-
may be used as a substrate, though with differing Vmax and Km.
mining glucose concentrations in the presence of tea polyphenols,
Both isozymes are competitively inhibited by their primary pro-
results from a hexokinase/G6PDH method was found to be in good
duct – G6P – furthering the need for a G6PDH coupled system
agreement with those from an HPLC with an RI (Refractive Index)
for glucose sensing (‘‘BRENDA; The Comprehensive Enzyme
detector. Comparatively poor results were obtained with the
Information System’’, 2014).
GOPOD assay, as the generated peroxide was likely non-specifically
quenched by the polyphenols, resulting in an artificially reduced
4.3. Glucose dehydrogenase
glucose concentration measurement (Xu, Leng, Wang, & Zhang,
2012). Other recent publications which incorporated use of a hex-
Three different glucose dehydrogenases, G6PDH (EC 1.1.1.49),
okinase/G6PDH assay include an assessment of glucose, sucrose
glucose-1-dehydrogenase (GDH, EC 1.1.1.47), and quinoprotein
and ethanol concentrations in fermenting wheat dough (Loveday
glucose dehydrogenase (PQQ-GDH, EC 1.1.5.2) are commonly used
& Winger, 2008) and a determination of the effects of controlled
in glucose sensing assays. A fourth glucose dehydrogenase type –
atmosphere storage on sugar concentrations and the activities of
FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) –
key sugar-modulating enzymes in apples (Zhu, Lui, Li, & Tian, 2013).
with promising characteristics was recently discovered
(Tsujimura et al., 2006).
4.4. Glucose 1-dehydrogenase
4.3.1. Glucose-6-phosphate dehydrogenase
GDH is a dimeric or tetrameric protein which, instead of utiliz-
G6PDH is a monomeric, dimeric, or tetrameric protein that cat-
ing an internal FAD cofactor to oxidize glucose, relies on the exter-
alyzes the second reaction of the pentose phosphate pathway
nal oxidative capacity of either NAD+ or NADP+.
(PPP):

Glucose-6-phosphate þ NADPþ ! 6-phospho-d-glucono-1; b-d-glucose þ NADðPÞþ ! d-glucono-1; 5-lactone þ NADPH ð6Þ

5-lactone þ NADPH ð4Þ While the majority of GDH isoforms are specific for D-glucose,
some – in particular those from Baccillus species – display greater
Unlike hexokinase, which catalyzes the reaction directly
activity when provided with 2-deoxy-D-glucose. Other sugars that
upstream in the PPP, G6PDH is not substrate promiscuous, and will
may be detectably oxidized include xylose, idose, and galactose.
accept only glucose-6-phosphate and 2-deoxy-D-glucose-6-phos-
Although the enzyme kinetics for GOx and GDH are comparable,
phate. The latter has been indicated thus far only for the human
wildtype GDH is rapidly inactivated at pHs and temperatures out-
and Thermotoga maritima isozymes, and results in a 90% reduction
side of physiological norms (Liang et al., 2013). For this reason, the
in activity as compared to glucose-6-phosphate (‘‘BRENDA; The
enzyme is not typically a first choice for incorporation into glu-
Comprehensive Enzyme Information System’’, 2014). Like hexoki-
cose-sensing assays; GDH does see some use though in handheld
nase, G6PDH is maximally active between pHs 7 and 9, with some
glucose meters for both food and medical applications (see below).
isoforms extending this range to pH 10. The enzyme may display
As per G6PDH, GDH activity may be followed spectrophotometri-
partial inhibition in the presence of ATP and NADPH (again, iso-
cally at 340 nm.
form dependent) and is broadly and strongly inhibited by copper,
cadmium, and mercury.
4.5. Quinoprotein glucose dehydrogenase
Because G6PDH is NADP+ dependent, its activity can be fol-
lowed spectrophotometrically at 340 nm. By conjugating the activ-
Quinoprotein glucose dehydrogenases, also known as pyrrolo-
ity of a hexokinase or glucokinase to that of G6PDH, the starting
quinoline quinine glucose dehydrogenases (PQQ-GDH), are a class
glucose concentration in a given solution can be back-calculated
of enzymes that utilize PQQ or ubiquinone as a redox cofactor for
via Beer’s Law:
the oxidation of glucose:
A ¼ eLc ð5Þ
d-glucose þ pyrroloquinoline quinine ! d-glucono-1;
where A = absorbance, e = molar absorptivity (extinction coefficient
5-lactone þ pyrroloquinoline quinol ð7Þ
in some literature), L is the path length of the cuvette (or other mea-
surement vessel) and c = concentration. As with any spectrophoto- PQQ-GDHs to date are only known to be synthesized in
metric method that utilizes NAD(P)H, care must be taken to prokaryotes. The enzymes in this family may be categorized into
ensure that the solution to be assessed does not already contain two distinct groups: soluble dimeric PQQ-GDHs (sPPQ-GDH) and
enzymes that are capable of independently evolving NADPH prior membrane-bound monomeric PQQ-GDHs (mPPQ-GDH). Of the
154 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
two groups mPPQ-GDHs display higher specificity for glucose rel-
ative to other sugars, but also require the presence of detergents
to ensure proper enzyme folding and cofactor retention. sPPQ-
GDHs are considerably less substrate specific: lactose, maltose,
allose, and several other sugars in addition to glucose will be
accepted by the active site with varying rates of turnover
(Stredanský, Monošík, Mastihuba, & Šturdík, 2013). Both groups
of enzymes require either Mg2+ or Ca2+ for catalytic activity and
are active across a fairly broad pH range (5–9); additionally,
because they do not use oxygen as a cofactor, both groups retain
maximal activity under anaerobic conditions. Regeneration of
PQQ in all cases can occur via interaction with other natural redox
molecules – such as ascorbate or NADPH – or artificial mediators
like phenazine methosulfate. Incorporation of a redox dye such
as DCPIP (A: 600 nm) as a mediator allows for spectrophotometric
determination of glucose concentration in an isolated system using
PQQ-GDH.
Despite their superior substrate specificity, mPQQ-GDHs have
seen little use in glucose sensing applications, due to the difficulty
associated with their expression and purification. Instead, most
efforts have focused on the incorporation and improvement of
their soluble counterparts. Wildtype sPQQ-GDHs have poor ther-
mal stability, reportedly losing 50% of their activity at 25 °C and
60–100% of their activity between 55 °C and 65 °C (Hofer,
Bönsch, Greiner-Stöffele, & Ballschmiter, 2011). To address this
and other deficiencies, recombinant sPQQ-GDHs which display
increased specificity for glucose and improved temperature toler-
ance have now been developed (Igarashi, Hirokawa, & Sode, 2004).
As compared to GOx, sPQQ-GDH turnover when using glucose
as a substrate is equivalent, and in many cases higher (3440/s for
Acinetobacter calcoaceticus versus 1890/s for A. niger).
Accordingly, sPQQ-GDH has seen widespread adoption in situations
where GOx’s requirement for O2 may preclude its use. Aside from
incorporation as an electrode in bio-fuel cells and glucose meters
(discussed below), the enzyme has been utilized within the realm
of food chemistry. For instance, Kurtinaitienė et al. (2010) demon-
strated the applicability of carbon paste-sPQQ-GDH electrodes for
measuring glucose concentration during wine yeast fermentation.
When placed under the same conditions in an online reactor, a
comparable carbon paste – GOx electrode rapidly ran out of free
oxygen and did not produce a signal that could be correlated with
glucose concentration.
4.6. FAD-glucose dehydrogenase
A relative late-comer in the field of glucose sensing, FAD-GDHs
utilize, as their name implies, an FADH cofactor to facilitate the
oxidation of glucose.
b-d-glucose þ ðFADÞGDH ! d-glucono-1;5-lactone þ ðFADH2 ÞGDH
ð8Þ
Mechanistically and structurally FAD-GDH is very similar to
GOx (Ferri, Kojima, & Sode, 2011). However, there is one important
difference: while GOx relies on O2 to regenerate its cofactor, FAD-
GDH does not and instead can use a variety of natural and intro-
duced mediators including an electron-transfer protein subunit
(bacterial FAD-GDH), ferricyanide, and 2,6-dichlorophenolin-
dophenol. As per PQQ-GDH, use of a redox dye as a mediator allows
glucose concentration to be determined spectrophotometrically in
an isolated system.
Like GOx, fungal FAD-GDH is highly specific for glucose, and dis-
plays virtually no activity when presented with alternate sub-
strates. On the other hand, the bacterial isoforms of the enzyme
are much less particular, and will accept maltose, rhamnose, and
xylose among other carbohydrates with varying rates (2.8–13%)
of activity relative to glucose. However, site-directed mutagenesis
of the active site of one bacterial FAD-GDH from Burkholderia cepa-
cia has successfully produced an enzyme that is specific for glucose
(Yamaoka, Yamashita, Ferri, & Sode, 2008).
Although a fungal FAD-GDH was identified as early as 1967
(Bak, 1967), interest in these enzymes for glucose sensing applica-
tions has been renewed only recently. Numerous configurations for
bio-fuel cells incorporating FAD-GDH and assorted mediators have
appeared in the literature (Milton, Giroud, Thumser, Minteer, &
Slade, 2014); however to date these systems have not seen direct
application in food-based systems.
5. Glucose meters
As the effect of diabetes on the sugar concentrations present in
urine have been known since ancient times, early efforts to develop
an all-in-one glucose quantification method focused on this bodily
fluid. A litmus-paper-like test that utilized glucose oxidase, perox-
idase, and a chromogen to quantify urine glucose was developed
by the Miles-Ames Laboratories in 1957. Because urine glucose
concentrations are dependent upon fluid intake however, a more
accurate means of establishing glycemic state was sought. In
1965, the first blood glucose test strip – the Dextrostix – was
developed at Miles-Ames; it too relied upon the activity of glucose
oxidase, and was widely adopted by the medical community. The
first glucose meters produced were cumbersome, but by 1974
technology had progressed sufficiently that Boehringer
Mannheim was able to market a meter for home use. Subsequent
developments in reflectance and electrochemistry-based detection
largely took the place of colorimetric glucose quantification, and
the use of alternate enzyme systems improved detection accuracy
in many cases.
Modern glucose meters, including the popular Accu-Check,
Precision, and Optimum lines, primarily rely on GOx, GDH, or
PQQ-GDH, with a small subset employing FAD-GDH for measure-
ment of blood glucose levels. The enzymes are directly impreg-
nated onto the test strips, and a small (1–10 lL) drop of fluid is
applied directly. The concentration determination is performed in
less than a minute, and based on either reflectance photometry
(colorimetric) or amperometry/coulometry (i.e. direct measure-
ment of electron current). Each detection system has strengths
and weaknesses, in part dictated by the specificity and stability
of the enzymes employed, and by the sensitivity and accuracy of
the meter. Overall, most handheld meters tend to be acceptably
accurate and precise, with intra-assay precision between 3% and
7% and a high correlation (r = 0.99) between a reference standard
and meter output (Pfützner et al., 2012).
Because handheld glucose meters are now mass-produced for
consumer market and may be purchased for far less than the cost
of an HPLC or CZE, their use is beginning to spread beyond the con-
fines of blood glucose testing. Results to date have been mixed,
with some studies finding that glucose meters, though functional,
were not ideally suited to their needs. For instance, an assessment
of glucose monitors for measuring residual glucose during wine-
making found that the meter employed was not sensitive to glu-
cose concentrations above 0.1%, a threshold indicative of the
potential for microbial contamination (Cook, Devlin, Ebeler, &
Butzke, 1998). Likewise, a canola protein fractionation study found
that, although capable of providing a reasonable free glucose esti-
mate, a One Touch glucose meter could not resolve whether the
glucose detected originated from glucosinolates or some other
source (Ser, Arntfield, Hydramaka, & Slominski, 2008). On a more
positive note, a glucose meter was used to monitor starch digestion
in vitro and found to provide comparable accuracy to spectropho-
tometric methods, with positive implications for future assessment
 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
of grain composition and quality (Sopade & Gidley, 2009). Most
promisingly, an assay has been developed for quantifying lactose
in dairy products, using a glucose meter to determine the amount
of glucose available in a sample after enzymatic hydrolysis
(Amamcharla & Metzger, 2011). That assay is currently being
tested in a commercial dairy facility, with an eye toward wide-
spread industry adoption (Morrison, 2014).
6. Non-enzymatic glucose sensors
While enzymatic glucose detection methods rely on the cat-
alytic activity of the enzymes such as GOx or GDH to oxidize glu-
cose, non-enzymatic glucose sensors (NEGS) forgo the use of
biological components entirely and instead utilize metal elec-
trodes. These electrodes can be made from a variety of elements
– copper, platinum, nickel, and gold being the most common –
and embodied as nanotubes, nanoparticles, porous films, or
assorted chemically modified surfaces. The sensitivity of these sys-
tems is quite good, with a general linear detection range of
104 mM to 1 mM of glucose, and many electrodes extend the
range by an order of magnitude or more in either direction
(Toghill & Compton, 2010). The effective sensitivity range
approaches or overlaps the average concentration of glucose in
blood (5 mM), but remains too low to be considered practical for
monitoring glucose concentration during the early stages of fer-
mentation for consumable or industrial ethanol production. Like
the early wet chemistry methods, NEGS also suffer from a lack of
sensitivity to glucose relative to other reducing sugars that may
be found in a complex food system. Additionally some species, par-
ticularly proteins and chloride ions, have a tendency to foul the
electrode, though this can be eliminated somewhat through the
use of pulsed amperometry or addition of surfactants to the solu-
tion of interest. As an alternative, it is possible to couple NEGS to
an upstream separation method, such as flow injection analysis
or liquid chromatography. This method allows for full separation
of all the species in a solution of interest, but in turn eliminates
the potential for real-time analysis of glucose concentration during
processing. Most electrodes capable of processing LC or FIA effluent
also have a reduced linear range, with a lower threshold of
103 mM being most common. It is likely that these and other
issues will be addressed as the field continues to grow: a literature
keyword search for NEGS shows that the field has expanded
rapidly from 2004 (0 papers) to the present, with more than 80
papers on the topic having been published in 2013 alone.
Although they are not yet ready to supersede enzyme- based or
HPLC-coupled methods for detecting glucose in complex solutions,
the adaptability of form factors coupled with sensitivity to low
concentrations of glucose may warrant future adoption of NEGS
for food chemistry applications.
7. HPLC
One of the most useful tools in the separation and identification
of chemical components of organic material is high performance
liquid chromatography (HPLC). The principle behind HPLC is the
analyte of interest is placed into a liquid mobile phase that carries
it through a column packed with a stationary phase. Separation of
the analyte is dependent on the type of chromatography being uti-
lized. The separation of carbohydrates and specifically glucose can
be achieved using multiple types of HPLC, including ion chro-
matography, reversed phase, hydrophilic interaction, and size
exclusion. HPLC’s versatility allows for the user to adjust the meth-
ods used to fit their chromatographic equipment.
Ion chromatography is perhaps the most commonly used form
of HPLC to separate monosaccharides. There are two types of ion
155
chromatography used: ion exchange and ion exclusion. Ion
exchange chromatography works by binding the analyte to the sta-
tionary phase inside the column, utilizing the ionic interaction
between oppositely charged molecules in a low concentration salt
solution. Once the materials are bound a gradient of increasing salt
concentration is used to begin separating the molecules. The
weaker the ionic interaction the earlier the interaction will termi-
nate and the analyte will elute from the column. The gradual
changing of the mobile phase pH can also be utilized to disengage
the analyte from the stationary phase. Since carbohydrates are typ-
ically negatively charged under basic conditions, anion-exchange
chromatography is employed as the separation mechanism. In
anion-exchange the pH is lowered in a gradient; as the pH lowers
the analyte becomes more protonated, and therefore neutral, ter-
minating the interaction. Alternately, the elution may be per-
formed isocratically with increasing concentrations of base. The
functional group on the stationary phase is typically a quaternary
amine or diethylaminoethyl (DEAE). The sample elution time rela-
tively long compared to other forms of chromatographic separa-
tion, but provides excellent resolution with high sensitivity. One
disadvantage of ion exchange chromatography is that high pH
ranges can cause epimerization or degradation of the sugars (Lee,
1990).
Ion exclusion is the second type of ion chromatography that
relies upon ion exchange columns to separate carbohydrates. The
technique separates molecular species based on their ability to
partition between the eluent that elutes and the eluent that
remains contained within the stationary phase resin. Cation-ex-
change columns are typically used with H+, Pb+2, Ca+2, or Na+ ions
bonded to the stationary phase. Molecules with similar charges are
repelled from the resin network, but neutral or oppositely charged
molecules can enter the network and are eluted based on either
their interaction with the ions or on size exclusion. This chromato-
graphic separation is typically done with an isocratic flow of a low
concentration of sulfuric acid or deionized water mobile phase. Ion
exclusion chromatography provides good separation for beverages
and foods that contain a large number of ionized compounds.
Separation of glucose from other components can be done very
quickly with good resolution. For example, glucose can be sepa-
rated from other sugars and organic acids in fruit juices in under
15 min (Kelebek, Selli, Canbas, & Cabaroglu, 2009).
Reversed phase (RP) is the most widely used form of HPLC and
has been used to separate monosaccharides (Zhang et al., 2013).
This technique uses a stationary phase with hydrophobic func-
tional groups attached with a polar mobile phase. The molecules
of interest interact with the hydrophobic groups on the stationary
phase and are eluted as the polarity of the mobile phase decreases.
The more hydrophobic the molecule is the longer it remains in
interaction with the column increasing its elution time. The mobile
phase typically consists of an organic solvent, such as acetonitrile,
the concentration of which is increased in a gradient throughout
the sample run. When RP-HPLC is used to separate monosaccha-
rides a C18 or C8 functional group is attached to the stationary
phase. In carbohydrate analysis by RP-HPLC the sugars are usually
derivatized with an aromatic group rendering them hydrophobic.
Due to the gradient nature of the separation RP-HPLC run times
are longer than some other forms of chromatography, but recent
advancements in column packing material has shortened the elu-
tion times (Bean, Ioerger, & Blackwell, 2011).
Hydrophilic interaction chromatography (HILIC) utilizes sta-
tionary phases that are polar – similar to normal phase HPLC –
but with mobile phases that are similar to those used in RP-
HPLC. The most common stationary phase for sugar analysis is a
silica particle with amino functional groups. The analytes are par-
titioned on the surface of the stationary phase and retained by
hydrogen bonding. Samples are separated based on the number
156 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
of polar groups and their configuration. The mobile phase can be a
gradient of decreasing organic solvent concentration or an isocratic
run of usually 70–80% acetonitrile. Glucose can be separated from
other sugars in under 15 min in HILIC. A more extensive review of
HILIC can be found by Jandera (2011).
Size exclusion chromatography (SEC) is commonly used to sep-
arate polymeric carbohydrates, but has been occasionally
employed to separate glucose. SEC separates molecules based on
their hydrodynamic volume and requires molecular weight differ-
ences of 10–20% to achieve baseline separation. Either an aqueous
or an organic solvent can be utilized as the mobile phase passing
through a polymer or silica bead stationary phase. Typically SEC
has relatively long run times and does have difficulty separating
glucose from fructose (Giannoccaro, Wang, & Chen, 2008).
The chromatographic separation is the first step in the analysis
of glucose and other sugars. Once the glucose is separated it can be
detected with several different types of detectors, including
Refractive Index (RID), Pulsed Amperometric (PAD), Evaporative
Light Scattering (ELSD), UV, and Fluorescence. The various detec-
tors are not all suited for each type of chromatography used, there-
fore selection of a detector needs to match the separation process.
The RID is considered a universal detector since it is measures
the difference in the refractive index of the sample eluent com-
pared to the reference cell. RID will work with both aqueous and
organic mobile phases. However, the measurement is sensitive to
the liquid contained in the reference cell; any changes to the
mobile phase creating differences to the reference cell liquid will
cause baseline shifts or movement. The detector is also sensitive
to changes in pressure and temperature. RID is very useful for
sugar detection since native sugars do not contain a chromophore
or fluorophore. Chromatographic separation techniques such as ion
exclusion and size exclusion are commonly paired with RID since
they operate with isocratic flow of the mobile phase. RID has been
used to detect glucose in many food and industrial systems.
Recently, Carballo, Zingarello, Maestre, Todolí, and Prats (2014)
used ion exclusion with RID to separate the sugars of oranges
and other citrus fruits, to quantify glucose they used a calibration
curve with a range of 100–5000 lg/L. A similar range was used
with sugars in grapevine berries (0.1–20 g/L) with an instrument
detection limit of 0.16 g/L (signal:noise = 3) (Eyéghé-Bickong,
Alexandersson, Gouws, Young, & Vivier, 2012). Glucose was
Fig. 3. Detection of monosaccharides from partially hydrolyzed and fermented citrus peel waste by HPAEC-PAD. Separation was carried out using a Dionex CarboPac PA-1
anion exchange column and a linear NaOH gradient. A Dionex ED50 PAD with gold electrode was used for monosaccharide detection. Reprinted with permission from
Widmer (2011).
separated by HILIC with RID from milk powders with a level of
detection at 29 lg/mL (Ma, Hou, Zhang, Wang, & He, 2014).
When paired with appropriate separation techniques, RID offers
good detection on sample materials known to contain reasonably
small amounts of glucose. RID does not offer the sensitivity of
PAD or ELSD, but it requires less sample preparation than fluores-
cence and provides better detection than UV.
The most common detector system used with ion exchange
chromatography is the PAD. Glucose and other carbohydrates are
detected by PAD via the measurement of the electric current gen-
erated by their oxidation on a gold or platinum electrode. This oxi-
dation at the surface of the electrode creates a residue on the
electrode which needs to be cleaned by increasing the volt poten-
tial which oxidizes the electrode and removes the carbohydrate
oxidation product. Then the potential is lowered to reduce back
to the original metal electrode. This utilization of three potentials
creates the pulse or waveform which continuously repeats over
the course of the sample analysis. PAD has been used to quantify
glucose and other sugars in many food and bioindustrial systems
including chickpeas (Gangola, Jaiswal, Khedikar, & Chibbar,
2014), biomass from oranges (Widmer, 2011) and corn stover
(Wang, Xu, Fan, Yong, & Yu, 2012) (Fig. 3). PAD has become widely
used due to its high sensitivity with a level of detection of 0.2 lM
for glucose (Gangola et al., 2014). Recent advancements in PAD for
carbohydrate analysis has been extensively reviewed by Rohrer,
Basumallick, and Hurum (2013).
ELSD is considered to be a universal detection system and is
commonly used to detect and quantify glucose as well as other car-
bohydrates. ELSD is a destructive detection technique that requires
the nebulization and evaporation of the mobile phase leaving
behind the analytes that are not evaporated. The analyte particles
enter the detection region and scatter light onto a photomultiplier
tube which measures the intensity as voltage. Since ELSD is com-
patible with many types of chromatographic separations used in
sugar analysis it has been used to quantify glucose in many food
systems ranging from milk, fruit and fruit juice, vegetables and cer-
eal grains (Ma et al., 2014; Shanmugavelan et al., 2013). ELSD offers
a reported level of detection of 0.37 lg/L for glucose. A disadvan-
tage to ELSD is that the calibration curve is not linear over large
concentrations of analyte. The detector is also susceptible to
mobile phase contaminants which can influence the signal:noise
 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
ratio and must be used with mobile phases that are volatile at the
temperature range needed for analysis of the sample.
Fluorescence and UV light detection can be used to detect sug-
ars but require more sample preparation steps or offer poor detec-
tion sensitivity compared to PAD or ELSD. Usually a derivatization
process with a chromophore or fluorophore is needed for detec-
tion; however some studies have been done with UV detection at
190–195 nm. The detectable threshold for glucose at that wave-
length range was found to be 9 lg/L (Shaw & Wilson, 1983). In
order to achieve detection levels approaching or even exceeding
PAD and ELSD the sugars need to be tagged with either a UV
absorbing chromophore, such as 1-phenyl-3-methyl-5-pyrazolone
(PMP) or a fluorophore, such as 2-(12-benzo[b}acridin-5(12H)-yl)-
acetohydrazide (BAAH). Dai et al. (2010) used RP-HPLC to separate
tagged monosaccharides, with glucose having a detection limit of
0.13 nmol. Fluorescence detection levels of 10 lg/L were found
after pre-column derivatization (Zhang et al., 2013). The detection
of glucose by UV or fluorescence detection is not as popular as the
other detection methods most likely due to the additional steps of
tagging the sugars which can cause alterations to chemical struc-
tures as well as being subject to tagging efficiency.
8. Capillary zone electrophoresis
Among analytical methods utilized for food-based analysis, cap-
illary zone electrophoresis (CZE) is one of the most diverse in its
applicability and widespread in its adoption. The instrumentation
can be used to separate not only carbohydrates (polymers, oligo-
mers and monomers) but also phenols, lipids, amino acids, nucleo-
tides, and assorted other molecules. Related as it is to gel
electrophoresis, CZE relies on differences in charge-based ionic
mobility to effect the separation of analytes from a complex mix-
ture. Capillaries most commonly are comprised of fused-silica,
the interior of which are negatively charged when exposed to a
background electrolyte (BGE) or pre-wash solution at a pH above
3. The composition of the BGE varies, and is dependent upon the
Fig. 4. Separation via CZE of 1. cellobiose, 3. galactose, 2. glucose, 5. rhamnose, 6. xylose, and 4. arabinose. Separation was carried out using either an Agilent 7100 or HP 3D
system coupled to a Diode Array Detector monitoring at 200 and 270 nm. Inset depicts the UV absorption spectra for glucose (dashes), arabinose (dots), and xylose (solid).
Structural information for photo-oxidized monosaccharides is provided in Oliver et al. (2014). Reprinted with permission from Oliver et al. (2013).
157
solubility and charge of the species to be analyzed, the method
of detection, etc. The capillaries themselves typically have a diam-
eter less than 100 lM, and a length of at least 30 cm; accordingly,
only a small amount of sample (on the order of nanoliters) is
required for each separation.
Because separation via CZE depends upon analyte charge, car-
bohydrates – which as a class of compounds are primarily neutral
– migrate poorly when unaided. There are several different
approaches which may be taken in order to mitigate this limita-
tion, including pre-column derivatization of carbohydrates with
an entity that is ionized in the desired BGE, complexation with
borate, and outright ionization of hydroxyl groups in a high pH
BGE (Oefner & Chiesa, 1994). Pre-column derivatization is advanta-
geous in that, in addition to altering the ionization profile of the
base molecules, it also provides an avenue for direct incorporation
of a fluorophore or chromophore. These conjugates may be then
directly detected via a coupled fluorometer or UV–VIS detector,
effectively solving two issues simultaneously.
For those samples that are not derivatized, either direct or indi-
rect detection via UV may still be used. At the traditional direct
detection absorbance wavelengths of 191–195 nm, the sensitivity
of CZE to most underivatized sugars, glucose included, is limited
to the mM range. However, researchers recently reported that
photo-oxidation of carbohydrates in an NaOH-based BGE allowed
for separation and quantification into the nm to lM range (7 lM
for glucose) at 270 nm (Oliver, Gaborieau, Hilder, & Castignolles,
2013; Sarazin, Delaunay, Costanza, Eudes, & Gareil, 2012). This
method has already been tested against orange juice, assorted
other juice samples and cognac (Rovio, Yli-Kauhaluoma, & Sirén,
2007), and is faster and more sensitive to carbohydrates than a
comparable HPLC-based method (Fig. 4). For indirect detection of
carbohydrates, a chromophore or fluorophore is added directly to
the BGE, and the elution of an analyte is detected as a temporary
drop in the background absorption or emission signal. As with
direct detection, indirect detection is several orders of magnitude
more sensitive than comparable HPLC methods; detection limits
158 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
as low as the pM range have been reported for glucose (Klockow,
Paulus, Figueiredo, Amado, & Widmer, 1994). Because indirect
detection requires a higher BGE signal than other methods, it is
more prone to baseline instability, which in turn affects calcula-
tions of analyze concentrations. Additionally, because the concen-
tration of BGE must be optimized such that migration of an analyte
through it is observable, the dynamic range of the method is lim-
ited to only a few orders of magnitude. Finally, inclusion of a chro-
mophore in the BGE presents an added difficulty for indirect
detection relative to direct detection simply because the chro-
mophore is one more component which must remain stable and
not complex with the analytes at the elevated pH used for analysis.
Still, the improved sensitivity that indirect detection provides con-
tinues to stimulate its use. Recently, Biluca and coworkers (2014)
assessed the fructose, sucrose, glucose and 5-hydroxymethylfur-
fural (5-HMF) content of 13 varieties of stingless bee honey using
indirect detection and a BGE comprised of sorbic acid, CTAB, and
NaOH (2014). The honey samples were found to contain a range
of carbohydrate concentrations, and to be less likely to form 5-
HMF after thermal treatment, with positive implications for the
honey’s commercial shelf life. For an extensive review on the
recent use of CZE for food based applications including carbohy-
drate analyses, the authors refer the reader to Garcıa-Cag ~ as,
Simo , Castro-Puyana, and Cifuentes (2014).
9. GC
Like CZE, gas chromatography (GC) is a general purpose
methodology that may be broadly applied to affect the separation
of many different types of compounds, among them fatty acids,
metal chelates, carbohydrates, and amino acids. First ideated by
Archer John Porter Martin in 1941, and formally presented by
Martin and A.T. James at a Biochemical Society Meeting in 1950,
the technique as originally developed relied upon an inert carrier
gas (usually nitrogen) to carry volatile analytes through a capillary
or tube that had been coated or packed with a stationary phase
possessing desirable characteristics (e.g. polarity). Separated via
their degree of interaction with the stationary phase, the analytes
were then passed to an appropriate detector, such as a titration
cell, a thermal conductivity sensor, or a gas density balance.
While the principle of the method remains unchanged, modern
GC systems have substituted nitrogen with helium or hydrogen
to improve separation efficiency, and are commonly coupled with
a mass spectrometer, flame ionization detector (FID), or nitrogen–
phosphorus detector (NPD).
Because GC necessitates that all analytes be volatile, carbohy-
drates, being primarily non-soluble in air, are poor candidates for
separation via this technique. Fortunately, a number of derivatiza-
tion methods have been developed, allowing monosaccharides and
oligosaccharides to be volatized, detected and quantified with high
resolution and replicability. For larger polysaccharides hydrolysis
can be carried out prior to derivatization to increase their volatility.
Common derivatizations for neutral sugars include the formation
of trimethylsilyl (TMS) ethers, TMS oximes, and aldononitrile acet-
ates. The formation of TMS ether derivatives is rapid (Sweeley,
Bentley, Makita, & Wells, 1963) and easily adapted to a host of
starting conditions. However, because D-glucose, for example,
may interconvert in solution between its a-gluco-furanose and b-
gluco-furanose forms, as well as between a-gluco-pyranose, b-
gluco-pyranose, and aldehyde forms as already discussed, TMS
ether derivatization produces a different end-product for each cyc-
lic tautomer and accordingly results in a very complex GC chro-
matogram. This problem is partially alleviated by converting the
sugars to oximes prior to the formation of TMS ethers, as the pos-
sible number of diastereoisomers is reduced to two. Overlap
between peaks is still possible however, especially when both glu-
cose and galactose are present within the same sample (Mason &
Slover, 1971). TMS oxime derivatization is advantageous in that
it produces an end-product that is stable for weeks under appropri-
ate storage conditions. For completely eliminating the multitude of
chromatogram peaks produced by diastereoisomers, alditol acetate
derivatization is a well established method. Each sugar is con-
verted from its aldose to alditol form and then acetylated, yielding
a single peak per monosaccharide. One major drawback of the
method in its unmodified form is an inability to differentiate
between ketose-aldose isomerization pairs: glucose for instance
may be reduced to glucitol, while fructose may be reduced to a
mixture of glucitol and mannitol. Additionally, the method does
not distinguish between glucose and natively present glucitol,
nor glucose and gulose. For food samples that are likely to contain
both glucose and fructose, ribose and ribulose, or another ketose–
aldose pair, alditol acetate derivatization may necessitate data cor-
roboration via a second quantization method. Recently, Brunton,
Gormley, and Murray (2007) determined the ratio by which fruc-
tose epimerizes either to mannose or glucose in potato extracts,
and used that information to calculate the fructose and glucose
concentrations for alditol acetate derivatized samples (2007).
Alternatively, in situations where glucose and fructose are present,
but glucitol and mannitol are not, sodium borohydride reduction
may be used prior to acetylation. This method may be further
adapted by the use of sodium borodeuteride instead of sodium
borohydride; in this case newly formed alditols will be deuterated
while previously existing ones will not be, allowing for differenti-
ation via MS (Fox, Morgan, & Gilbert, 1988). Many other derivatiza-
tion options to fill a variety of different experimental requirements
exist; for a more thorough treatment than what is presented here,
see Orato (2012).
A number of different detectors may be coupled to a GC system
for reliable identification and quantification of glucose and other
carbohydrates. As mentioned previously, common general purpose
detection methods include FID, MS, and NPD. Of the three, NPD is
the least well suited to detection of neutral sugars, as they do not
typically contain nitrogen or phosphorous atoms; the detector may
be used however to quantify amino and iminosugars when they
are present in a sample. FID makes use of a hydrogen-air flame
to combust analytes as they elute from the GC column; it is partic-
ularly sensitive to hydrocarbons and has a dynamic range of three
orders of magnitude at the electrode (Hinshaw, 2006). Using alditol
acetate derivatization and GC-FID, Montoyo-Arroyo and coworkers
(2014) were able to quantify a number of sugars in pectic fractions
from purple pitaya, including fucose at a concentration of
0.06 ± 0.01 g/100 g dry weight in an alkali soluble fraction (2014).
Because mass spectrometry and in particular tandem mass spec-
trometry can provide information about an analyte’s mass and
likely structure, coupling an MS to a GC allows for identification
of most peaks in a manner independent of comparisons of elution
times between samples and standards. In particular, MS is useful
in situations where multiple analytes, diastereoisomers excepted,
are eluting as a single peak, an event that is not readily apparent
with other detection methods. The limit of detection (LOD) for
GC–MS depends upon the monitoring mode, the number of quad-
rupoles, and of course the samples to be analyzed: Wang, Wang,
and Cai (2013) report LODs ranging from mg/g to pg/g for pesti-
cides and other contaminants in a variety of food systems (2013).
Recently, Uri, Juhász, Polgár, and Bánfalvi (2014) used GC–MS (pos-
itive mode) to obtain a metabolic profile – encompassing sugars,
sugar acids, fatty acids, amino acids, etc. – of commercial potato
cultivars throughout their development and storage (2014).
Sugars were derivatized via a TMS method, and the concentrations
of three – glucose, fructose, and sucrose – were found to decrease
considerably in tubers over a 15-week storage period.
 A.L. Galant et al. / Food Chemistry 188 (2015) 149–160
10. Other methods
Beyond those techniques described in the preceding sections,
several other methods for detecting and quantifying glucose and
other carbohydrates have been developed. In particular, various
forms of fourier transform (FT) spectroscopy, (Barnaba &
Mencarelli, 2014) and FT-NIR (Chen, Danao, Singh, & Brown,
2014), as well as 1H NMR (Cao et al., 2014) have been applied with
good success against a variety of food matrixes. Much like MS,
these methods have the capacity to provide both quantitative
and structural information relating to a sample. Additionally, IR
and NMR are both non-destructive processes, allowing the same
sample to be analyzed repeatedly with no loss of substance. For
analyses that require chromatographic separation of a complex
mixture, IR systems can be incorporated downstream of a GC or
LC. Although less sensitive than GC- or LC-MS, IR-based detection
can provide complimentary, and in many cases more complete,
structural information about an analyte under investigation
(Visser, 2002). Several reviews have been published on the applica-
tion of these methods to food systems; for more information read-
ers are encouraged to refer to Rodriguez-Saona and Allendorf
(2011); or Marcone et al. (2013).
11. Conclusions
Clearly glucose can be considered one of the more important
molecules whether we are discussing metabolic processes or food
systems. Detection and quantification has been studied for literally
centuries, with diabetes research leading to many significant find-
ings. Diabetes will continue to drive this research field with an
emphasis on non-invasive/non-destructive detection of blood glu-
cose. However, testing any new methodology requires checking
results against known standard protocols, thus scientists will con-
tinue to use and enhance the current enzymatic and non-enzy-
matic methodologies discussed in this review for many years to
come.
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