Accepted Manuscript

Title: Rapid Quantitative Detection of Glucose Content in

Glucose Injection by Reaction Headspace Gas
Chromatography

Authors: Wei-Qi Xie, Yi-Xian Gong, Kong-Xian Yu

PII: S0021-9673(17)31338-9
DOI: http://dx.doi.org/10.1016/j.chroma.2017.09.018
Reference: CHROMA 358847

To appear in: Journal of Chromatography A

Received date: 18-4-2017

Revised date: 2-8-2017
Accepted date: 6-9-2017

Please cite this article as: Wei-Qi Xie, Yi-Xian Gong, Kong-Xian Yu,
Rapid Quantitative Detection of Glucose Content in Glucose Injection
by Reaction Headspace Gas Chromatography, Journal of Chromatography
Ahttp://dx.doi.org/10.1016/j.chroma.2017.09.018

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Rapid Quantitative Detection of Glucose Content in Glucose Injection by
Reaction Headspace Gas Chromatography

Wei-Qi Xie,1,2* Yi-Xian Gong,1 and Kong-Xian Yu3

1.
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

Guangzhou, China
2.
School of Materials Science and Engineering, South China University of Technology,

Guangzhou, China
3.
Health Supervision Bureau of Liaoning Province, Shenyang, China

*Corresponding author. Tel/fax: +86-15913171507; E-mail: mswqxie@mail.scut.edu.cn (Wei-Qi

Xie).

Highlights

 Rapid quantifying the glucose content in glucose injection.

 This method is based on the oxidation reaction between glucose in glucose
injection and potassium dichromate under the acidic conditions.
 The carbon dioxide (CO2) formed from the oxidation reaction can be
quantitatively detected by gas chromatography.
 The HS-GC method is rapid, practical and can be utilized for quantitatively
analyzing the glucose content in glucose injection related applications.

Abstract: This work investigates an automated method for rapid quantifying the
glucose content in glucose injection by reaction headspace gas chromatography
(HS-GC). This technique is based on the oxidation reaction of glucose in glucose
injection with potassium dichromate under the acidic conditions. The carbon dioxide
(CO2) formed from the oxidation reaction can be quantitatively detected by GC. The
results show that the conversion of glucose in glucose injection can be achieved under
the given conditions (at 85 oC for 20 min), the relative standard deviation (RSD) of
1
this HS-GC technique in the glucose determination was within 2.91%, and the
measured glucose contents in glucose injection samples closely match those measured
by the reference technique (relative differences < 6.45%). The new HS-GC technique
is rapid, practical and can be used to the batch detection of the glucose content in
glucose injection related applications.
Keywords: Glucose content; glucose injection; Headspace gas chromatography;
Carbon dioxide

1. Introduction
Glucose is a simple sugar with the molecular formula C6H12O6 and is the most
important energy source for human body [1-3]. Glucose injection is a parenteral
aqueous solution containing glucose for intravenous administration, which is usually
used to provide the energy for patients who are inconvenient to eat [4-6]. Therefore, it
is highly desired for rapid and automated analytical technique for the detection of
glucose content to monitor the quality of glucose injection.
Traditionally, titration method is the commonly used technique for determining
glucose content in glucose injection [7,8]. In this method, the glucose sample is first
reacted with sodium hypoiodite (NaIO). After the complete reaction between the
aldehyde in glucose sample and NaIO, the unreacted NaIO is then oxidized to sodium
iodate (NaIO3) under an alkaline condition. Afterward, NaIO3 is titrated with sodium
thiosulfate (Na2S2O3), and thus the glucose content in glucose injection can be
calculated. Obviously, the procedures for the titration are very complicated and
time-consuming, especially when conducting the batch measurements.
There are also some analytical techniques that can be utilized for the glucose
content quantification based on instruments, e.g., nuclear magnetic resonance (NMR),
Spectrophotometer, Fourier transform infrared spectroscopy (FT-IR) and polarimeter
[9-13]. The major drawbacks in these techniques are the accuracy and precision in the
glucose content determination, especially for the samples with complicated matrix.
The negative impacts from the co-existing compounds are difficult to be eliminated.
Moreover, time-consuming and complicated pretreatment must be done before
2
conducting some of these techniques.
Headspace gas chromatography (HS-GC) is an efficient technique for analyzing
volatile substances in different kinds of complex matrices [14-16]. It is based on the
vapor-liquid equilibrium of a volatile species in a nonvolatile matrix in a closed
headspace vial. Because direct liquid phase detection is not necessary, HS-GC
technique avoids the sample matrix effect on the analysis. HS-GC can also be used to
analyze some non-volatile species that could be quantitatively converted to their
related volatile substances by some reactions [17,18]. In an earlier research [19], we
successfully developed a reaction HS-GC technique to quantitative detect isocyanate
groups in the organic intermediates, in which by detecting the carbon dioxide (CO2)
formed from the chemical reaction between isocyanate groups and water, the
isocyanate groups in the organic intermediates can be determined. According to the
literatures [20], the glucose can be quantitatively converted to CO2 by the oxidation
reaction between glucose and potassium dichromate (K2Cr2O7) under the acidic
conditions, which can be written as
2  3
C 6 H 12 O 6  4 Cr 2 O 7  32 H  6 CO 2
 8 Cr  22 H 2 O (1)

Since Eq. (1) can be easily conducted in a closed headspace vial, it is possible
that the glucose content quantification can be realized by analyzing the CO2 generated
from Eq. (1). Moreover, the batch analysis of the glucose content in glucose injection
could be realized by utilizing the auto-headspace samplers in the HS-GC system.
The objective of the present work was to investigate a reaction HS-GC technique
to rapidly quantify the glucose content in glucose injection. The main efforts in this
study were directed at the optimization of the reaction conditions (i.e., reaction
temperature/time, dosage of K2Cr2O7 and acid concentration) for the complete glucose
conversion. The performance (i.e., accuracy and precision) of the new HS-GC
technique were also examined during the glucose analysis.

2. Materials and methods

2.1. Chemicals

3
 All chemicals (i.e., potassium dichromate and sulfuric acid) were analytical
grade and obtained from a chemical supplier (Shanghai Macklin Co., Ltd. (Shanghai,
China). A series of glucose standard solutions were prepared by adding the original
glucose samples to appropriate amount of distilled water. The glucose injection
samples (5 - 50% glucose) were purchased from hospitals and suppliers in southern
China.
2.2. Instrument and operations
All testing were conducted by using an automatic headspace sampler (Thermo
HS TriPlus 300, US) connected to a GC system (Agilent GC 7890A, US) equipped
with a thermal conductivity detector (TCD) and a GS-Q capillary column (length = 30
m, inner diameter = 0.32 mm with thickness of stationary phase = 1 μm), operating at
a temperature of 105 °C with nitrogen carrier gas with a constant flow of 2.7 mL/ min.
The operating procedures for headspace auto-sampler consisted of 20 min of high
shaking at 85 °C to allow sample equilibration; pressurization pressure = 1.00 bar;
carrier gas pressure = 1.50 bar; vial pressurization time = 15 s; sample loop fill time =
10 s; and transfer time = 20 s; sample loop volume = 3 mL.
2.3. Procedure of sample preparation
A 100 μL of glucose injection sample was first was added into a headspace vial
(20 mL). Afterward, 200 μL of K2Cr2O7 solution (0.40 mol/L) and 200 μL of H2SO4
(8.0 mol/L) was then added into the vial, which was immediately sealed by rubber
septa. After that, the headspace vials were then placed into the headspace sampler and
automatically analyzed by HS-GC.

3. Results and discussion

3.1. Dosage of K2Cr2O7
In this study, the given volume of glucose injection was 100 μL. On the basis of
Eq. (1), an excess amount of K2Cr2O7 is needed to achieve a complete conversion of
glucose to CO2. As shown in Fig. 1, a complete conversion equilibrium can be
obtained by adding 80.0 μmol of K2Cr2O7 under the given conditions. In this research,
200 μL of K2Cr2O7 solution (0.40 mol/L), equals to 80.0 μmol, was selected in the
4
reaction.
3. 2. Effect of acidity
On the basis of Eq. (1), hydrogen ion is required for achieving the completeness
of glucose conversion under the given conditions. Fig. 2 shows the effect of acidity
(sulfuric acid concentration) on the CO2 formation from the oxidation reaction of
glucose, and it can be observed that the sulfuric acid concentration should not be less
than 8.0 mol/L. Hence, 8.0 mol/L of sulfuric acid (equals to 16.0 mol/L hydrogen ion)
was used in this research to ensure the completeness of the glucose conversion.
3. 3. Reaction (Equilibration) temperature and time
In general, higher temperature is helpful to accelerate the reaction rate and
minimize the CO2 dissolution in the solution. Fig. 3 shows the normalized GC peak
area for CO2 after a 30 min reaction time between inorganic carbon species and
hydrochloric acid at different temperatures (e.g., 45, 55, 65, 75, 85 and 95 oC), from
which we can see that a complete reaction can be achieved at the temperatures above
85 oC. Hence, 85 oC was selected as the reaction (equilibration) temperature in the
present research to ensure the complete reaction.
Fig. 4 shows the time effect on the GC peak area of CO2 formed from Eq. (1). It
can be seen that 20 min is needed for the complete conversion of glucose to CO2.
Therefore, 20 min was used as the headspace equilibration time in the following
studies.
3.4. Chromatogram from the HS-GC testing
According to Eq. (1), CO2 is the sole volatile compound formed from this
reaction. The headspace measurement is based on the vapor-liquid equilibrium of CO2
in the sealed headspace vial, and thus the total amount of glucose in the headspace
vial can be indirectly quantified by the GC detection of the CO2 formed from Eq. (1).
Fig. 5 (a) shows a chromatogram from the headspace measurement on a typical
glucose injection sample under the given GC column conditions. It can be seen that
CO2 can be well separated from other volatile substances (i.e., O2) in the HS-GC
testing, indicating the inertness and stability of the given GC column conditions for
CO2 analysis. In this method, the CO2 in the vapor phase of headspace vial could be
5
the interference. However, their influences can be easily minimized by conducting
blank testing (see Fig. 5 (b)).

3.5. Method calibration

An external standard curve can be adopted to calibrate this new HS-GC
technique. This calibration was achieved by adding known amounts of glucose
samples in a series of headspace vials that contain sulfuric acid and K2Cr2O7 solutions.
These headspace sample vials were then quantitatively analyzed by HS-GC after the
oxidation reaction of glucose. According to the data from the HS-GC analysis of these
standard samples, a linear external standard curve was obtained, i.e.,

A  - 0 . 08 (  0 . 23 )  289 . 5 (  1 . 1 4)  n (n =7, R2 = 0.9992) (2)

or

A  a ( Δ a )  s( Δ s)  n (2-1)

where A represents the peak area of the CO2 formed from Eq. (1), n represents the
number of moles of the glucose in sample (in mmol), a, ∆a, s, ∆s, are the intercept,
uncertainty of the intercept, the slope and uncertainty of the slope, in Eq. (2-1),
respectively.
On the basis of Eqs. (1) and (2), the glucose content in glucose injection can be
calculated by

ms nM ( A  a)M
C   
s
(3)
V V sV

where C, Ms, ms and m represent the glucose content in glucose injection, the
molar mass of glucose (180 g/mol), and the weight of glucose and the volume of
glucose injection sample, respectively.
The limit of quantification (LOQ) for the number of moles of glucose can be
obtained from the equation below (Eq. (4)), which is 7.94×10-3 mmol (equals to
1.43 % for the glucose content in glucose injection). It meets the requirements for the
glucose content analysis in glucose injection.

6
 10  a
LOQ  (4)
s

3.6. Method performance

3.6.1. Method precision
The precision (i.e., repeatability) testing of this method was studied by the
quintuplicate measurements of glucose content for three glucose injection samples.
The results obtained from the measurements show that the relative standard deviation
(RSD) of the glucose determination is within 2.91% (as shown in Table 1), in which
the errors are from the uncertainties in sample preparation, sampling and HS-GC
analysis.
3.6.2. Method validation
The accuracy of this new HS-GC technique was examined by conducting a
recovery test. The known amounts of glucose injection samples were spiked into the
sample solutions. After the solutions reacted with K2Cr2O7 under the acidic conditions,
they were then quantitatively detected by HS-GC according to procedures mentioned
in the experimental section. The glucose content in these samples can be obtained on
the basis of Eqs. (1)-(4), as showed in Table 2. It can be seen that the recoveries in
these measurements ranged from 97.5% to 102%, indicating that this HS-GC
technique is reliable to be used in the quantitative detection of glucose content in
glucose injection.
The HS-GC technique was also assessed by comparing the data obtained from
the HS-GC technique and the reference titration technique, based on 15 glucose
injection samples. As listed in Table 3, the data of the glucose content in these
samples analyzed by the HS-GC technique has a good correlation with the data from
the reference titration technique and the relative differences (RD) between the data
obtained from the new HS-GC technique and the titration method are less than 6.45%.
Since the new method is efficient and reliable, it can be a potential tool for
quantitatively detecting the glucose content in glucose injection related research.

7
4. Conclusions
A new reaction HS-GC technique for rapidly determining the glucose content in
glucose injection has been investigated. Compared with the traditional titration
method, this new method is much simpler and more efficient. The results indicate that
the present method is precise, automated and accurate. With the headspace
auto-sampler and GC system, the present technique can carry out automated testing
that is appropriate for the batch analysis of glucose content to efficiently monitor the
quality of glucose injection. Future work may focus on eliminating the possible
interfering substances in the samples.

Acknowledgements
The authors acknowledge the financial support from the State Key Laboratory of
Pulp and Paper Engineering, South China University of Technology, China.

8
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10
Figure Captures

Fig. 1 Effect of K2Cr2O7 dosage on the glucose conversion

Fig. 2 Effect of the concentration of sulfuric acid on the glucose conversion

11
Fig. 3 Effect of reaction (Equilibration) temperature on the CO2 equilibration

Fig. 4 Effect of reaction (Equilibration) time on the CO2 equilibration

12
Fig. 5 (a) Chromatogram of a glucose sample from HS-GC testing

Fig. 5 (b) Chromatogram of a blank sample from HS-GC testing

13
Table 1 The repeatability of the method

Glucose content, %
Replica no.
Sample 1 Sample 2 Sample 3

1 10.29 21.24 29.24

2 10.12 20.58 29.27

3 9.97 21.52 28.82

4 10.16 20.19 29.97

Average 10.14 20.88 29.08

RSD, % 1.30 2.91 2.47

Table 2 Recovery test

Sample ID Glucose content, % Recovery

Added Measured

1 5.35 5.42 101

2 10.29 10.03 97.5

3 13.82 13.72 99.3

4 3.57 2.51 98.3

5 8.36 8.49 102

14
Table 3 Comparison of the methods on glucose content quantification

Glucose content, % Relative difference, %

Sample ID
Present method Ref. method*

1 5.47 5.35 2.24

2 15.19 15.42 -1.49

3 20.68 20.36 1.57

4 30.52 31.02 -1.61

5 25.14 25.56 -1.64

6 4.13 4.35 -5.06

7 19.47 18.98 2.58

8 24.68 24.52 0.65

9 50.24 49.07 2.38

10 49.13 49.52 -0.79

11 51.16 51..69 -1.03

12 26.31 26.72 -1.53

13 41.52 40.35 2.90

14 11.23 10.55 6.45

15 16.94 17.14 -1.17

* The reference titration method

15