# Detection of Gelatin Adulteration in Traditional Chinese Medicine: Analysis of Deer-Horn Glue by Rapid-Resolution Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Authors: Jia Chen; Xian-Long Cheng; Feng Wei; Qian-Qian Zhang; Ming-Hua Li; Shuang-Cheng Ma
Journal: Journal of Analytical Methods in Chemistry
 (2015)

---

## Abstract

Simultaneous identification of donkey-hide gelatin and bovine-hide gelatin in deer-horn glue was established by rapid-resolution liquid chromatography-triple quadrupole mass spectrometry. Water containing 1% NH4HCO3 was used for sample dissolution and trypsin was used for hydrolysis of the gelatins. After separation by a SB-C18 reversed-phase analytical column, collagen marker peptides were detected by mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The method was specific, precise and reliable, and suitable for detection of adulterants derived from donkey-hide gelatin and bovine-hide gelatin in deer-horn glue.


---

## Body

## 1. Introduction

Deer-horn glue (Cervi Cornus Colla) is a traditional Chinese medicine (TCM) that has been widely used in China for about 2000 years. It is a solid glue prepared from deer horn by decoction and concentration [1]. It is viewed as a nutritious, high-quality TCM, as indicated in “Shennong’s Herbal,” and is predominantly used for treating kidney disorders and Qi deficiency. It is claimed that long-time consumption of deer-horn glue will nourish yin, replenish blood, and prolong life. Because of the high market price and an inability to satisfy demand, adulteration is common and the most widely practiced approach is to substitute and/or replace the authentic material with donkey- and bovine-hide gelatin.It has long been difficult to control the quality of deer-horn glue because of the absence of appropriate quality assessment methods. The polymerase chain reaction method has been used in DNA analysis for collagen identification [2, 3], but the method is not suitable for gelatin identification because of the breakdown of gelatin DNA during sample processing. Literature research has revealed that proteomic methods have been proposed as alternative tools for the assessment of collagen species in gelatins [4] and mass spectrometry has been successfully applied to elucidate differences among homological gelatins [5]. In our work, the focus of research has been on method specificity for differentiation of homological gelatins. In our previous work [6, 7], for instance, tryptic peptides of gelatins were measured by ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and principal component analysis was used to classify donkey-hide gelatin, bovine-hide gelatin, and deer-horn glue. Thereafter, gelatins were analyzed by doubly charged selected ion monitoring (DCSIM) with tandem mass spectrometry (MS/MS) to aid in the identification of the gelatins. The possibility of detecting the target peptides in such gelatins with rapid-resolution liquid chromatography (RRLC) coupled to electrospray ionization- (ESI-) ion trap (IT) MS would be a useful development.Generally, HPLC-QQQ MS/MS is a sensitive analytical method available for detection of the adulterants. As shown recently, high-pressure liquid chromatography-mass spectrometry (HPLC-MS) is a widely used technique for qualitative and quantitative analyses, combining the efficient separation capability of HPLC with the powerful structural capability of MS [8–19]. In addition, the MS method offers the potential for high sensitivity and selectivity through multiple reaction monitoring (MRM) without the need for baseline chromatographic separation of the target analytes [20–22].In the present work, RRLC-QQQ-MS with MRM has been used for characterization of deer-horn glue and detection of gelatin adulteration. A fully validated method has been developed, permitting measurement of the collagen marker peptides in commercial samples of deer-horn glue adulterated with donkey-hide and bovine-hide gelatins.


## 2. Experimental

### 2.1. Materials and Reagents

Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA) and HPLC-grade acetonitrile (MeCN) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrahigh-purity water was prepared using a Milli-Q water purification system (Millipore Corporation, Bedford, MA, USA). Trypsin (sequencing grade) was obtained from Promega (Madison, WI, USA). Syringe filters (0.22μm) were purchased from Millipore (Billerica, MA, USA). All other chemicals used were of analytical grade. All samples were collected by the National Institute for Food and Drug Control.


### 2.2. Sample Preparation

First, 100 mg of the gelatin was dissolved in 50 mL of a 1% NH4HCO3solution (pH 8.0). Then 10 μL of trypsin solution (1 mg/mL in 1% NH4HCO3, pH 8.0) was added to 100 μL of the gelatin solution. The mixture was incubated at 37°C for 12 h. All gelatin samples were prepared in this way. The sources of the gelatin samples are shown in Table 1.Table 1
Gelatin sources.
Sample
Standard gelatin
Source
Lot number by NIFDC
1
Deer-horn glue
Cervus elaphus Linnaeus
121694-201301
2
Donkey-hide gelatin
Equus asinus L.
121274-201202
3
Bovine-hide gelatin
Bos taurusdomesticus Gmelin
121695-201301


### 2.3. Chromatographic Separation and Mass Spectrometry

The RRLC analysis was performed using an Agilent 1200 LC system (Agilent, MA, USA). Chromatographic separation was performed on an Agilent Zorbax SB-C18 reversed-phase analytical column (100 mm × 2.1 mm; 1.8μm particle size) at a column temperature of 45°C. The sample injection volume was 5 μL. The mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile (eluent B). Gradient elution was performed as follows: 0–25 min eluent B 5% → 20%; 25–40 min eluent B 20% → 50%. The flow rate was 0.3 mL·min−1.Mass spectrometry experiments were performed with an ESI source in positive ion mode. The vaporizer temperature was maintained at 350°C. The temperature of the drying gas was set at 350°C. The flow rate of the drying gas and the pressure of the nebulizer gas were set at 6 L/min and 60 psi, respectively. In MRM scan mode, the precursor and product ions should be set. The intensity of the precursor ion should be higher after optimizing the fragmentation voltage and the intensity of the product ion should also be higher after collision energy (CE) optimization. After optimization, the voltages for fragmentation and the CE were recorded (Table2). An Agilent ChemStation was used for instrument (Agilent 6410B series triple quadrupole MS system) control and data processing. This included definitive identification of metabolites using retention times and fragmentation transition matching. Chromatographic separation was achieved using identical conditions to those described above for IT-MS experiments [6, 7]. Gradient elution was performed as follows: 0–25 min eluent B 5% → 20%; 25–40 min eluent B 20% → 50%. The flow rate was 0.3 mL·min−1.Table 2
Precursor and product ions for the gelatin species and operating parameters for fragmentation voltage and collision-activated dissociation voltage.
Number
Precursor
Product ion
Originated from
Retention time
Fragment voltage
Collision energy
m
/
z
m
/
z
min
eV
eV
A1
732.8
817.9/961.9
Deer-horn glue
11.2080
175
30
A2
765.4
554.0/733.0
Deer-horn glue
17.1209
135
15
B1
641.3
783.3/726.2
Bovine-hide gelatin
7.4309
135
37
B2
790.9
912.4/841.3
Bovine-hide gelatin
12.5446
175
32
B3
747.3
903.3/847.1
Bovine-hide gelatin
13.4004
155
26
B4
604.8
569.8/910.1
Bovine-hide gelatin
15.2002
135
25
C1
618.8
721.9/778.9
Donkey-hide gelatin
7.7407
135
23
C2
539.8
612.4/923.8
Donkey-hide gelatin
10.1043
135
15
C3
765.9
823.1/991.0
Donkey-hide gelatin
18.8379
155
45


## 2.1. Materials and Reagents

Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA) and HPLC-grade acetonitrile (MeCN) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrahigh-purity water was prepared using a Milli-Q water purification system (Millipore Corporation, Bedford, MA, USA). Trypsin (sequencing grade) was obtained from Promega (Madison, WI, USA). Syringe filters (0.22μm) were purchased from Millipore (Billerica, MA, USA). All other chemicals used were of analytical grade. All samples were collected by the National Institute for Food and Drug Control.


## 2.2. Sample Preparation

First, 100 mg of the gelatin was dissolved in 50 mL of a 1% NH4HCO3solution (pH 8.0). Then 10 μL of trypsin solution (1 mg/mL in 1% NH4HCO3, pH 8.0) was added to 100 μL of the gelatin solution. The mixture was incubated at 37°C for 12 h. All gelatin samples were prepared in this way. The sources of the gelatin samples are shown in Table 1.Table 1
Gelatin sources.
Sample
Standard gelatin
Source
Lot number by NIFDC
1
Deer-horn glue
Cervus elaphus Linnaeus
121694-201301
2
Donkey-hide gelatin
Equus asinus L.
121274-201202
3
Bovine-hide gelatin
Bos taurusdomesticus Gmelin
121695-201301


## 2.3. Chromatographic Separation and Mass Spectrometry

The RRLC analysis was performed using an Agilent 1200 LC system (Agilent, MA, USA). Chromatographic separation was performed on an Agilent Zorbax SB-C18 reversed-phase analytical column (100 mm × 2.1 mm; 1.8μm particle size) at a column temperature of 45°C. The sample injection volume was 5 μL. The mobile phase consisted of 0.1% formic acid in water (eluent A) and acetonitrile (eluent B). Gradient elution was performed as follows: 0–25 min eluent B 5% → 20%; 25–40 min eluent B 20% → 50%. The flow rate was 0.3 mL·min−1.Mass spectrometry experiments were performed with an ESI source in positive ion mode. The vaporizer temperature was maintained at 350°C. The temperature of the drying gas was set at 350°C. The flow rate of the drying gas and the pressure of the nebulizer gas were set at 6 L/min and 60 psi, respectively. In MRM scan mode, the precursor and product ions should be set. The intensity of the precursor ion should be higher after optimizing the fragmentation voltage and the intensity of the product ion should also be higher after collision energy (CE) optimization. After optimization, the voltages for fragmentation and the CE were recorded (Table2). An Agilent ChemStation was used for instrument (Agilent 6410B series triple quadrupole MS system) control and data processing. This included definitive identification of metabolites using retention times and fragmentation transition matching. Chromatographic separation was achieved using identical conditions to those described above for IT-MS experiments [6, 7]. Gradient elution was performed as follows: 0–25 min eluent B 5% → 20%; 25–40 min eluent B 20% → 50%. The flow rate was 0.3 mL·min−1.Table 2
Precursor and product ions for the gelatin species and operating parameters for fragmentation voltage and collision-activated dissociation voltage.
Number
Precursor
Product ion
Originated from
Retention time
Fragment voltage
Collision energy
m
/
z
m
/
z
min
eV
eV
A1
732.8
817.9/961.9
Deer-horn glue
11.2080
175
30
A2
765.4
554.0/733.0
Deer-horn glue
17.1209
135
15
B1
641.3
783.3/726.2
Bovine-hide gelatin
7.4309
135
37
B2
790.9
912.4/841.3
Bovine-hide gelatin
12.5446
175
32
B3
747.3
903.3/847.1
Bovine-hide gelatin
13.4004
155
26
B4
604.8
569.8/910.1
Bovine-hide gelatin
15.2002
135
25
C1
618.8
721.9/778.9
Donkey-hide gelatin
7.7407
135
23
C2
539.8
612.4/923.8
Donkey-hide gelatin
10.1043
135
15
C3
765.9
823.1/991.0
Donkey-hide gelatin
18.8379
155
45


## 3. Results and Discussion

Method validation was performed according to the guidelines of the Chinese Pharmacopoeia (2010 edition) for TCM. The key performance parameters evaluated were selectivity, signal linearity, sensitivity, and repeatability.


### 3.1. Selectivity

The specificity of the method was investigated using deer-horn glue as a blank sample, while donkey- and bovine-hide gelatin serving as positive control samples. In previous work, the gelatins were characterized using DCSI-MS/MS. In this study, doubly charged ions atm/z 641.3, 747.5, 790.9, and 604.8, which are the species-specific peptides of bovine-hide gelatin, were selected for monitoring. Also, the fragments of these monitored ions resulted in the following additional characteristic molecular ion pairs:m/z 641.3 → 783.3, 641.3 → 726.2, 747.5 → 903.3, 747.5 → 847.1, 790.9 → 912.4, 790.9→841.3, 604.8→569.8, and 604.8→910.1. Doubly charged ions at 539.8, 618.8, and 765.9, which are species-specific peptides of donkey-hide gelatin, were selected for monitoring and yielded the following molecular ion transition pairs: 539.8 → 612.4, 539.8 → 923.8, 618.8 → 721.9, 618.8 → 778.9, 765.9 → 823.1, and 765.9 → 991.0. The chromatographic peaks were verified by checking the retention times and fragments of the peaks. As a result, chromatographic peaks for deer-horn glue were different to those of donkey-hide gelatin and bovine-hide gelatin. This meant that the mass spectra for the peptides in deer-horn glue were not subject to interference, as shown in Figure 1.Figure 1
(a) Characteristic selected ion chromatograms for deer-horn glue. (b) Characteristic selected ion chromatograms for bovine-hide gelatin. (c) Characteristic selected ion chromatograms for donkey-hide gelatin.
(a)
(b)
(c)


### 3.2. Signal Linearity

#### 3.2.1. Calibration Curves for Bovine-Hide Gelatin

A matrix solution of deer-horn gelatin standard was prepared by dissolving 100.0 mg of standard in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Next, 100.6 mg of the bovine-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Increasing aliquots (0.1, 0.5, 1.0, 1.5, and 5.0 mL) of the bovine-hide gelatin standard solutions were dissolved in 10 mL of the differing matrix solutions. Then, 100 μL of the gelatin standard solution was taken and 10 μL of trypsin solution (1 mg/mL in 1% NH4HCO3, pH 8.0) was added. The mixtures were incubated at 37°C for 12 h.


#### 3.2.2. Calibration Curves for Donkey-Hide Gelatin

For sample preparation, 119.6 mg of the donkey-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). This solution was then subjected to the same method as outlined in Section 3.2.1.The regression equations, correlation coefficients, and test ranges for calibration are shown in Table3. The results showed that there was an excellent correlation between the ratio of peak area response and concentration for each compound within the test ranges examined.Table 3
Signal linearity curves for two analytes.
Analytes
Linear equations
Range (μg/mL)
R
2
Bovine-hide gelatin
Y
=
3715
X
+
321.1
20.12–1006
0.957
Donkey-hide gelatin
Y
=
32485
X
-
1130
23.92–1196
0.995


### 3.3. Sensitivity

The limit of detection (LOD), defined as the peak signal with a signal to noise ratio = 3/1, was determined based on injections (2μL) of low level standard solutions. The results demonstrated that the method was very sensitive with LODs of 10 × 10−6g/mL and 20 × 10−6g/mL for the peptides in the bovine- and donkey-hide gelatin samples, respectively.


### 3.4. Repeatability

Five replicate samples were prepared by the above method and the selected ion chromatograms, shown in Figures2 and 3, confirm that the method provided reproducible detection of the collagen marker peptides.Figure 2
Characteristic selected ion chromatograms obtained for the tryptic digests of five donkey-hide gelatins.Figure 3
Characteristic selected ion chromatograms obtained for the tryptic digests of six bovine-hide gelatins.


### 3.5. Species Identification by RRLC-QQQ-MS

The complex peptide pools obtained by tryptic digestion of the gelatins were subjected to LC-MS/MS and the characteristic molecular ion peaks for the bovine- and donkey-hide gelatins were detected as ion pairs listed in Table2. Typical MRM chromatograms are shown in Figures 4 and 5. Commercial samples were positively identified after matching specific peptides in these samples with the corresponding reference samples. In 29 commercial samples of deer-horn glue analyzed, 12 tested positive for bovine-hide gelatin and 2 tested positive for donkey-hide gelatin, as indicated in Table 4. Overall, the proposed method provides a new and efficient route for unambiguous measurement of collagen marker peptides of bovine- and donkey-hide gelatins.Table 4
Results for commercial samples of deer-horn glue.
Number
Sample
Number
Origin
Donkey-hide gelatin
Bovine-hide gelatin
Deer-horn glue
1
Deer-horn glue
001
Henan Province
−
+
+
2
Deer-horn glue
002
Henan Province
−
+
−
3
Deer-horn glue
003
Shandong Province
−
+
−
4
Deer-horn glue
004
Henan Province
−
+
+
5
Deer-horn glue
005
Hubei Province
−
+
−
6
Deer-horn glue
006
Hunan Province
−
+
+
7
Deer-horn glue
007
Hebei Province
+
−
+
8
Deer-horn glue
008
Hebei Province
+
−
+
9
Deer-horn glue
009
Hunan Province
−
+
+
10
Deer-horn glue
010
Henan Province
−
+
+
11
Deer-horn glue
011
Henan Province
−
+
−
12
Deer-horn glue
012
Hunan Province
−
+
+
13
Deer-horn glue
013
Inner Mongolia Autonomous Region
−
+
+
14
Deer-horn glue
014
Shandong Province
−
+
+
15
Deer-horn glue
015
Shandong Province
−
−
+
16
Deer-horn glue
016
Shandong Province
−
−
+
17
Deer-horn glue
017
Beijing Municipality
−
−
+
18
Deer-horn glue
018
Beijing Municipality
−
−
+
19
Deer-horn glue
019
Beijing Municipality
−
−
+
20
Deer-horn glue
020
Hubei Province
−
−
+
21
Deer-horn glue
021
Hubei Province
−
−
+
22
Deer-horn glue
022
Hubei Province
−
−
+
23
Deer-horn glue
023
Henan Province
−
−
+
24
Deer-horn glue
024
Henan Province
−
−
+
25
Deer-horn glue
025
Henan Province
−
−
+
26
Deer-horn glue
026
Shandong Province
−
−
+
27
Deer-horn glue
027
Shandong Province
−
−
+
28
Deer-horn glue
028
Beijing Municipality
−
−
+
29
Deer-horn glue
029
Beijing Municipality
−
−
+
30
Deer-horn glue
121694-201301
Standard gelatin from NIFDC
−
−
+
31
Donkey-hide gelatin
121274-201202
Standard gelatin from NIFDC
+
−
−
32
Bovine-hide gelatin
121695-201301
Standard gelatin from NIFDC
−
+
−Figure 4
Representative MRM chromatograms for bovine-hide gelatins in tested samples.Figure 5
Representative MRM chromatograms for donkey-hide gelatins in tested samples.


## 3.1. Selectivity

The specificity of the method was investigated using deer-horn glue as a blank sample, while donkey- and bovine-hide gelatin serving as positive control samples. In previous work, the gelatins were characterized using DCSI-MS/MS. In this study, doubly charged ions atm/z 641.3, 747.5, 790.9, and 604.8, which are the species-specific peptides of bovine-hide gelatin, were selected for monitoring. Also, the fragments of these monitored ions resulted in the following additional characteristic molecular ion pairs:m/z 641.3 → 783.3, 641.3 → 726.2, 747.5 → 903.3, 747.5 → 847.1, 790.9 → 912.4, 790.9→841.3, 604.8→569.8, and 604.8→910.1. Doubly charged ions at 539.8, 618.8, and 765.9, which are species-specific peptides of donkey-hide gelatin, were selected for monitoring and yielded the following molecular ion transition pairs: 539.8 → 612.4, 539.8 → 923.8, 618.8 → 721.9, 618.8 → 778.9, 765.9 → 823.1, and 765.9 → 991.0. The chromatographic peaks were verified by checking the retention times and fragments of the peaks. As a result, chromatographic peaks for deer-horn glue were different to those of donkey-hide gelatin and bovine-hide gelatin. This meant that the mass spectra for the peptides in deer-horn glue were not subject to interference, as shown in Figure 1.Figure 1
(a) Characteristic selected ion chromatograms for deer-horn glue. (b) Characteristic selected ion chromatograms for bovine-hide gelatin. (c) Characteristic selected ion chromatograms for donkey-hide gelatin.
(a)
(b)
(c)


## 3.2. Signal Linearity

### 3.2.1. Calibration Curves for Bovine-Hide Gelatin

A matrix solution of deer-horn gelatin standard was prepared by dissolving 100.0 mg of standard in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Next, 100.6 mg of the bovine-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Increasing aliquots (0.1, 0.5, 1.0, 1.5, and 5.0 mL) of the bovine-hide gelatin standard solutions were dissolved in 10 mL of the differing matrix solutions. Then, 100 μL of the gelatin standard solution was taken and 10 μL of trypsin solution (1 mg/mL in 1% NH4HCO3, pH 8.0) was added. The mixtures were incubated at 37°C for 12 h.


### 3.2.2. Calibration Curves for Donkey-Hide Gelatin

For sample preparation, 119.6 mg of the donkey-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). This solution was then subjected to the same method as outlined in Section 3.2.1.The regression equations, correlation coefficients, and test ranges for calibration are shown in Table3. The results showed that there was an excellent correlation between the ratio of peak area response and concentration for each compound within the test ranges examined.Table 3
Signal linearity curves for two analytes.
Analytes
Linear equations
Range (μg/mL)
R
2
Bovine-hide gelatin
Y
=
3715
X
+
321.1
20.12–1006
0.957
Donkey-hide gelatin
Y
=
32485
X
-
1130
23.92–1196
0.995


## 3.2.1. Calibration Curves for Bovine-Hide Gelatin

A matrix solution of deer-horn gelatin standard was prepared by dissolving 100.0 mg of standard in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Next, 100.6 mg of the bovine-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). Increasing aliquots (0.1, 0.5, 1.0, 1.5, and 5.0 mL) of the bovine-hide gelatin standard solutions were dissolved in 10 mL of the differing matrix solutions. Then, 100 μL of the gelatin standard solution was taken and 10 μL of trypsin solution (1 mg/mL in 1% NH4HCO3, pH 8.0) was added. The mixtures were incubated at 37°C for 12 h.


## 3.2.2. Calibration Curves for Donkey-Hide Gelatin

For sample preparation, 119.6 mg of the donkey-hide gelatin standard was dissolved in 50 mL of a 1% NH4HCO3 solution (pH 8.0). This solution was then subjected to the same method as outlined in Section 3.2.1.The regression equations, correlation coefficients, and test ranges for calibration are shown in Table3. The results showed that there was an excellent correlation between the ratio of peak area response and concentration for each compound within the test ranges examined.Table 3
Signal linearity curves for two analytes.
Analytes
Linear equations
Range (μg/mL)
R
2
Bovine-hide gelatin
Y
=
3715
X
+
321.1
20.12–1006
0.957
Donkey-hide gelatin
Y
=
32485
X
-
1130
23.92–1196
0.995


## 3.3. Sensitivity

The limit of detection (LOD), defined as the peak signal with a signal to noise ratio = 3/1, was determined based on injections (2μL) of low level standard solutions. The results demonstrated that the method was very sensitive with LODs of 10 × 10−6g/mL and 20 × 10−6g/mL for the peptides in the bovine- and donkey-hide gelatin samples, respectively.


## 3.4. Repeatability

Five replicate samples were prepared by the above method and the selected ion chromatograms, shown in Figures2 and 3, confirm that the method provided reproducible detection of the collagen marker peptides.Figure 2
Characteristic selected ion chromatograms obtained for the tryptic digests of five donkey-hide gelatins.Figure 3
Characteristic selected ion chromatograms obtained for the tryptic digests of six bovine-hide gelatins.


## 3.5. Species Identification by RRLC-QQQ-MS

The complex peptide pools obtained by tryptic digestion of the gelatins were subjected to LC-MS/MS and the characteristic molecular ion peaks for the bovine- and donkey-hide gelatins were detected as ion pairs listed in Table2. Typical MRM chromatograms are shown in Figures 4 and 5. Commercial samples were positively identified after matching specific peptides in these samples with the corresponding reference samples. In 29 commercial samples of deer-horn glue analyzed, 12 tested positive for bovine-hide gelatin and 2 tested positive for donkey-hide gelatin, as indicated in Table 4. Overall, the proposed method provides a new and efficient route for unambiguous measurement of collagen marker peptides of bovine- and donkey-hide gelatins.Table 4
Results for commercial samples of deer-horn glue.
Number
Sample
Number
Origin
Donkey-hide gelatin
Bovine-hide gelatin
Deer-horn glue
1
Deer-horn glue
001
Henan Province
−
+
+
2
Deer-horn glue
002
Henan Province
−
+
−
3
Deer-horn glue
003
Shandong Province
−
+
−
4
Deer-horn glue
004
Henan Province
−
+
+
5
Deer-horn glue
005
Hubei Province
−
+
−
6
Deer-horn glue
006
Hunan Province
−
+
+
7
Deer-horn glue
007
Hebei Province
+
−
+
8
Deer-horn glue
008
Hebei Province
+
−
+
9
Deer-horn glue
009
Hunan Province
−
+
+
10
Deer-horn glue
010
Henan Province
−
+
+
11
Deer-horn glue
011
Henan Province
−
+
−
12
Deer-horn glue
012
Hunan Province
−
+
+
13
Deer-horn glue
013
Inner Mongolia Autonomous Region
−
+
+
14
Deer-horn glue
014
Shandong Province
−
+
+
15
Deer-horn glue
015
Shandong Province
−
−
+
16
Deer-horn glue
016
Shandong Province
−
−
+
17
Deer-horn glue
017
Beijing Municipality
−
−
+
18
Deer-horn glue
018
Beijing Municipality
−
−
+
19
Deer-horn glue
019
Beijing Municipality
−
−
+
20
Deer-horn glue
020
Hubei Province
−
−
+
21
Deer-horn glue
021
Hubei Province
−
−
+
22
Deer-horn glue
022
Hubei Province
−
−
+
23
Deer-horn glue
023
Henan Province
−
−
+
24
Deer-horn glue
024
Henan Province
−
−
+
25
Deer-horn glue
025
Henan Province
−
−
+
26
Deer-horn glue
026
Shandong Province
−
−
+
27
Deer-horn glue
027
Shandong Province
−
−
+
28
Deer-horn glue
028
Beijing Municipality
−
−
+
29
Deer-horn glue
029
Beijing Municipality
−
−
+
30
Deer-horn glue
121694-201301
Standard gelatin from NIFDC
−
−
+
31
Donkey-hide gelatin
121274-201202
Standard gelatin from NIFDC
+
−
−
32
Bovine-hide gelatin
121695-201301
Standard gelatin from NIFDC
−
+
−Figure 4
Representative MRM chromatograms for bovine-hide gelatins in tested samples.Figure 5
Representative MRM chromatograms for donkey-hide gelatins in tested samples.


## 4. Conclusions

The RRLC-MS method with MRM provides an excellent qualitative tool for quality assessment of deer-horn glue because of its high sensitivity and specificity. As shown, collagen marker peptides associated with donkey-hide gelatin and bovine-hide gelatin and presented as adulterants in deer-horn glue, were readily detected. Furthermore, according to the signal linearity, we can estimate the amount of adulteration roughly and provide a specified limitation for adulteration. In survey analysis, almost 50% of commercial samples were found to have been adulterated by the addition of donkey- and/or bovine-hide gelatin, which were more than 3% of adulterants in samples according to the signal linearity.



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*Source: 259757-2015-10-04.xml*