Food Chemistry 173 (2015) 619–623

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

Analytical Methods

Fast and sensitive collagen quantification by alkaline hydrolysis/

hydroxyproline assay
Cassia Maria Lins da Silva a, Eliani Spinelli b, Silvana Vianna Rodrigues a,⇑
a
Instituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista S/N, Centro, Niterói, RJ 24020-150, Brazil
b
Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mario Viana 523, Niterói, RJ 24241-000, Brazil

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

Article history: A preparative protein alkaline hydrolysis procedure, as part of a spectrophotometric collagen quantifica-
Received 24 April 2013 tion method, is presented. The procedure is suitable for small amounts of fresh solid or liquid samples.
Received in revised form 9 March 2014 Various aspects of the procedure, such as the NaOH concentration, time needed to hydrolyse different
Accepted 14 October 2014
collagen contents, buffer strength of the reagent solution, pH control of the hydrolysate and spectropho-
Available online 22 October 2014
tometric conditions, were evaluated. Compared to other procedures that use alkaline hydrolysis, the sen-
sitivity of this procedure was increased by a factor of 5. Compared to the conventionally used Association
Keywords:
of Official Analytical Chemists (AOAC) acid hydrolysis method, the reaction time was reduced from 16 h
Collagen
Alkaline hydrolysis
to 40 min and the amount of sample from 4 g to 3–20 mg, producing equivalent results when applied to
Hydroxyproline porcine liver and sausage samples.
Protein Ó 2014 Elsevier Ltd. All rights reserved.
Spectrophotometry

1. Introduction with fluorometric detection was applied to determine the amount

Collagen is the most important connective-tissue protein. It is
mainly present in skin, bones, tendons, cartilages and teeth. In
the food industry, this protein is widely used to improve the elas-
ticity, consistency and stability of foods (Santana, Sato, & da Cunha,
2012) in a wide variety of products, such as drinks, soups, noodles,
candies and meat products (Taffin & Pluvinet, 2006). Collagen
hydrolysates have also acquired importance in protein supple-
ments, where they have been shown to be useful in maintaining
the nitrogen balance in older people (Hays, Kim, Wells,
Kajkenova, & Evans, 2009). The analysis of collagen can be an aid
in meat authentication because different amounts of collagen are
present in different meat cuts (Ballin, 2010). Collagen content
has also been used as an index of the quality for meat sausages
(Messia, Di Falco, Panfili, & Marconi, 2008).
Many techniques to estimate collagen have been described.
Capillary electrophoresis was used to detect fraud associated with
the addition of collagen to milk and dairy products to increase the
apparent protein content of the product (Dong et al., 2012). Ion
exchange high-performance chromatography with pulsed ampero-
metric detection (HPIEC-PAD) associated with microwave-assisted
hydrolysis of collagen was used to analyse meat products (Messia
et al., 2008). High-performance liquid chromatography (HPLC)
⇑ Corresponding author. Tel.: +55 21 26292140; fax: +55 21 26292143.
E-mail address: silvana@vm.uff.br (S.V. Rodrigues).
of collagen in meat and meat products (Vázquez-Ortíz, Morón-
Fuenmayor, & González-Méndez, 2004) after hydrolysis of the
samples using the AOAC (Association of Official Analytical Chem-
ists) procedure (AOAC, 2000). However, these techniques require
expensive equipment and costly maintenance.
The most widely used method for the quantification of collagen
is acid hydrolysis followed by a colorimetric hydroxyproline assay
(AOAC, 2000; Hofman, Hall, Cleaver, & Marshall, 2011; Kolar, 1990;
Neuman & Logan, 1950), which represents 12–14% w/w of collagen
(Edwards, & O’Brien, 1980; Hofman et al., 2011; Ignat’eva et al.,
2007). In this procedure, the imino acid undergoes oxidation due
to the action of chloramine-T and becomes a pyrrole, which then
reacts with 4-dimethylaminobenzaldehyde, known as Ehrlich’s
reagent (Kolar, 1990; Stegemann & Stalder, 1967), generating a
substance that absorbs in the visible region of the spectrum. The
acid hydrolysis of collagen is generally performed with the addi-
tion of 6 mol L 1 HCl under heat in diverse experimental condi-
tions (Table 1). The major disadvantage of these processes is the
extended time required to complete the hydrolysis even for a small
quantity of sample.
Alkaline hydrolysis is used to digest a wide variety of substrates
to analyse the amino acid composition of proteins, especially in the
determination of tryptophan, which is stable under basic condi-
tions (Fountoulakis & Lahm, 1998). Collagen hydrolysis under alka-
line conditions is faster and more intense than hydrolysis in an
acidic medium (Chirita, 2000). However, conditions for sensitive

http://dx.doi.org/10.1016/j.foodchem.2014.10.073
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
620 C.M.L. da Silva et al. / Food Chemistry 173 (2015) 619–623
Table 1
Summary of the data from studies where collagen was hydrolysed in acid pH.
References Hydrolysis conditions
Acid Pressure (kPa) Temperature (°C)
1
Neuman and Logan (1950) 6 Mol L HCl 344.5 – 3
1
Stegemann and Stalder (1967) 6 Mol L HCl – 107
1
Edwards and O’Brien (1980) 6 Mol L HCl 210 120
1
Kolar (1990) 3.5 Mol L H2SO4 – 105
1
Vázquez-Ortíz et al. (2004) 6 Mol L HCl – 150
and reproducible determination in fresh solid samples are not well
established. Huszar, Maiacco, and Naftolin (1980) replaced acid
with alkaline hydrolysis for dehydrated tissue samples, which
were autoclaved at 120 °C; under the proposed working condi-
tions, the reaction time was reduced to 10 min. Reddy and
Enwemeka (1996) adapted this procedure for samples of cold
dehydrated tissue and used a reaction time of 20 min. Hofman
et al. (2011), using liquid and freeze-dried samples with high col-
lagen content, failed to reproduce the results of Reddy and
Enwemeka (1996) and had to conduct alkaline hydrolysis for 3 h
to complete the sample digestion; there was a decrease in recovery
with the increase in collagen concentration, most likely because
the hydrolysis was incomplete. As the colorimetric assay is pH-
sensitive, a buffer was usually added to the hydrolysate, assuming
that the buffer had enough capacity to bring the solution to the
proper pH, although this may not be the fact when dealing with
very strong alkaline solutions.
The objective of this study was to develop a simple, fast, sensi-
tive and reproducible procedure for the determination of collagen,
which is applicable to fresh solid samples such as tissues, meat and
meat products and to collagen solutions, without the use of expen-
sive resources, which would be accessible to any laboratory with
basic instrumentation. The parameters involved in the alkaline
hydrolysis of collagen, such as the extent of hydrolysis and the con-
centration of NaOH, were explored. The sensitivity of the colori-
metric determination, using hydroxyproline, was optimised for
alkaline hydrolysates. Finally, the accuracy of the method was
evaluated by comparison with the official AOAC method for por-
cine liver and sausages.
2. Experimental
2.1. Apparatus
The absorbances were recorded with a visible spectrophotome-
ter (model SP-22, Biospectro, Curitiba, Brazil) with 1-cm optical
path glass cuvettes. The adjustments and measurements of pH
were carried out with a pH metre (model HI 3222, Hanna Instru-
ments, USA). Reagents and samples were weighed either with a
0.1-mg precision analytical balance (model AUW220D, Shimadzu,
Japan) or with a 0.001-g precision balance (model BG 400,
GEHAKA, São Paulo, Brazil), depending on the application. The
homogenisation of solutions was performed with a vortex (model
Lab Danger, IKA, Staufen, Germany). A thermostatic bath (New
Technique, Brazil) was employed for the colorimetric reaction. An
autoclave (model 415/1, FANEM, Brazil) and high-temperature
12-mL polypropylene screw-capped tubes were used for alkaline
hydrolysis. Acid hydrolysis was conducted in Erlenmeyer flasks
in an oven (model 315 SE, FANEM, Brazil).
Treatment of hydrolysate
Time (h)
pH neutralization
16 Evaporation to dryness, followed by addition
of 2 mL citrate–acetate buffer pH 6.
2–4 Overnight evaporation to dryness under vacuum,
followed by addition of citrate–acetate buffer pH 6–6.5
16 Dilution
6 Evaporation to dryness, followed by addition
of 2.0 mL 0.6 Mol L 1 sodium citrate buffer, pH 2.2.
2.2. Reagents and solutions
The standard collagen solutions were obtained from a
4 mg mL 1 type I collagen solution in 20 mmol L 1 acetic acid
(Sigma–Aldrich, USA, Cat. No. C3867). Dilutions of this solution
were prepared in 20 mmol L 1 acetic acid at ratios of 1:10–
1:12.5. The stock solution of 600 mg mL 1 hydroxyproline
(Sigma–Aldrich, USA, Cat. No. H54409) was prepared in ultrapure
water. To construct the standard curve, dilutions were prepared
from stock solutions in the range of 0.12–12 mg mL 1.
Chloramine-T (Cat. No. 402869), 4-dimethylaminobenzalde-
hyde (Cat. No. 156477), sodium acetate trihydrate (Cat. No
32318), 1-propanol (Cat. No. 402893) from Sigma–Aldrich, USA
and perchloric acid (Vetec, Brazil, Cat. No. 909), anhydrous citric
acid (Spectrum, USA, Cat. No. C1280), 2-propanol (Tedia, USA,
Cat. No. PS-2032.4) and sulphuric acid (Vetec, Brazil, Cat. No.
190) were used in the preparation of reagents for the colorimetric
determination of hydroxyproline (Kolar, 1990; Reddy, &
Enwemeka, 1996). A 50% w/v stock solution of NaOH (Vetec, Brazil,
Cat. No. 101) was used for sample preparation.
2.3. Adjustment of the analytical conditions for the colorimetric
determination of hydroxyproline in alkaline hydrolysates
2.3.1. Determination of the maximum absorbance wavelength (max)
Absorbance spectra were recorded for 8 lg mL 1 hydroxypro-
line solutions in water (according to the AOAC method) and in
2 mol L 1 NaOH (as reported by Reddy and Enwemeka (1996)),
and kmax was determined.
2.3.2. Effect of pH on the sensitivity of the hydroxyproline assay, using
standard solutions
An 8 lg mL 1 hydroxyproline solution was prepared either in
2 mol L 1 NaOH, following Reddy and Enwemeka (1996) or in
water. To 0.5 mL of the standard solution, 4.5 mL of the buffered
chloramine-T solution was added and the pH of the reaction med-
ium was measured. After 25 min of reaction, 5 mL Ehrlich’s reagent
was added and allowed to react for 15 min at 65 °C. Reagents and
proportions followed the conditions established in Reddy and
Enwemeka (1996). The absorbances were measured at kmax.
2.3.3. Effect of pH on the sensitivity of the hydroxyproline assay using
alkaline hydrolysates
Neutralised and non-neutralised alkaline collagen hydrolysates
were assayed. The hydrolysates were obtained under the following
conditions: 500 lL of 2 mol L 1 NaOH were added to 200 lL of col-
lagen standard solution (143 lg collagen, 17.8 lg hydroxypro-
line); hydrolysis was performed in 20 min in an autoclave at
120 °C. After the reaction, the hydrolysate was acidified with
 C.M.L. da Silva et al. / Food Chemistry 173 (2015) 619–623
500 lL of 1 mol L 1 H2SO4 solution and pure water was added to
achieve a final volume of 10 mL for photometric quantification. A
parallel experiment was conducted in which the neutralisation
was omitted. The hydroxyproline assay was then performed under
the conditions described in Section 2.3.2.
2.3.4. Analytical curve
Standard solutions (n = 4) of hydroxyproline in water were pre-
pared in the following concentrations: 0.12; 0.30; 0.60; 1.2; 2.4;
3.0; 3.6; 4.2 and 4.8 lg mL 1. The limits of detection (LOD) and
quantification (LOQ) were calculated according to Miller and
Miller (1993), using an Excel worksheet.
2.4. Optimisation of alkaline hydrolysis conditions
2.4.1. Study of the influence of the concentration of NaOH on the
efficiency of the hydrolysis
The amount of collagen was kept constant (383.3 lg). A con-
stant volume of 500 lL of 2, 4, 8, 10 and 12.5 mol L 1 NaOH
(n = 4 in each case) was added, and hydrolysis was performed as
stated in Section 2.3.3. Recovery was calculated assuming 12.5%
(w/w) hydroxyproline in collagen (Kolar, 1990).
2.4.2. Study of the duration of hydrolysis
Four replicate hydrolysis experiments were conducted with 10,
20, 30, 40, 50 and 60 min reaction time. The study was repeated for
different amounts of collagen (71.37, 142.74, 285.47 and
378.18 lg). In each case, 500 lL of 7 mol L 1 NaOH was added.
Recovery was calculated as described in Section 2.4.1.
2.5. Comparison of the method with acid hydrolysis using the AOAC
method 990.26
The proposed method was applied to porcine sausages and liver
acquired from the local market, and compared with AOAC method
990.26 (AOAC, 2000). Samples (200 g) were homogenised in a food
processor. For the AOAC method, 5 aliquots of 4 g of the homoge-
nate were transferred to an Erlenmeyer flask and hydrolysed with
30 mL of 3.5 mol L 1 H2SO4 at 105 ± 1 °C for 16 h. The hydrolysate
was transferred to a 500-mL volumetric flask, filled with ultrapure
water and then filtered. The amount of hydroxyproline was mea-
sured spectrophotometrically.
For alkaline hydrolysis, 3–7 mg of sausage and 10–20 mg of
liver were used; 5 replicates were analysed. The amount of sample
was calculated based on an approximate content (100 lg collagen)
to avoid the need for further dilution in the hydroxyproline assay.
The alkaline hydrolysis took place in 12-mL polypropylene screw-
capped tubes, in which 0.5 mL of 7 mol L 1 NaOH was added. The
tubes were placed in the autoclave for 40 min. The hydrolysis tem-
perature in the autoclave was approximately 120 °C (p  98.1 kPa).
The hydrolysate was acidified with 0.5 mL 3.5 mol L 1 H2SO4,
transferred to a 10-mL volumetric flask and filled with ultrapure
water.
Table 2
Effect of the pH of the reaction medium in the determination of hydroxyproline (n = 4) in hydroxyproline standard solution (8 lg mL
hydroxyproline); k = 560 nm.
Standard solution In NaOH
In ultrapure water
Hydrolysate Without acidification
With acidification
621
2.6. Statistical analysis
Student’s paired t-test at 95% confidence level (Excel software)
was used to compare the results obtained by acid and alkaline
hydrolysis of samples, as described in Section 2.5.
3. Results and discussion
3.1. Colorimetric determination of hydroxyproline for alkaline
hydrolysates
Mitchell and Taylor (1970) tested 23 spectrophotometric meth-
ods for the determination of hydroxyproline and found some
authors worked at wavelengths that were not at the maximum
and justified neither this nor other changes in analytical condi-
tions. AOAC method 990.26 recommends the use of 558 ± 2 nm.
Although the determination by Reddy and Enwemeka (1996) was
performed at 550 nm, our verification of the analytical wavelength
revealed kmax at 562 ± 2 nm, which was then chosen for all subse-
quent experiments. No difference in kmax was observed for
hydroxyproline solutions in ultrapure water or in 2 mol L 1 NaOH.
Because published methods (Hofman et al., 2011; Reddy, &
Enwemeka, 1996) performed the spectrometric hydroxyproline
assay after alkaline hydrolysis without the prior neutralisation of
the hydrolysate, we investigated a possible effect of pH on the sen-
sitivity of the method, keeping the proportions and concentrations
of reagents consistent with those described by Reddy and
Enwemeka (1996). We found that the buffer capacity of the chlora-
mine T reagent was not strong enough to neutralise the alkaline
medium (Table 2). A strong decrease in sensitivity was observed
when the absorbances in neutral and alkaline mediums were com-
pared for assays performed with standard solutions of hydroxypro-
line. When the hydroxyproline assay was performed with
hydrolysates, the same effect was observed (Table 2). Acidification
before the addition of chloramine-T proved to be a necessary step,
and the hydrolysates were, therefore, acidified in subsequent
experiments. In fact, the formation of the pyrrole resulting from
the reaction between chloramine-T and hydroxyproline only gave
a good yield when the pH was approximately neutral
(Stegemann & Stalder, 1967). Based on our results, sensitivity
was sacrificed in previous works to reduce the number of steps,
i.e., to allow the direct use of the alkaline hydrolysate. In the pres-
ent work, both neutralised and non-neutralised hydrolysates were
diluted to a final volume of 10 mL to match the analytical curve.
Even under these conditions, sensitivity was impaired for non-
acidified hydrolysates (Table 2). Curiously, although Hofman
et al. (2011) did not adopt the prior neutralisation of the hydroly-
sate, they observed that 1:50 dilutions of the hydrolysate led to
more reproducible results. Dilution most likely decreased the pH
and favoured the colorimetric reaction. Additionally, procedures
that involve acid hydrolysis, as reported in the literature, include
dilutions of the hydrolysate, which is sometimes neutralised or
brought to dryness (Edwards & O’Brien, 1980; Kolar, 1990;
Neuman, & Logan, 1950; Reddy & Enwemeka, 1996; Stegemann
& Stalder, 1967).
1 1
) and collagen hydrolysate (1.8 lg mL
Addition of buffered Chloramine-T Absorbance ± S.D.
pH before pH after
>14 13.5 0.351 ± 0.016
– 6.3 1.767 ± 0.205
13.1 12.8 0.057 ± 0.003
3.0 6.0 0.313 ± 0.004
622 C.M.L. da Silva et al. / Food Chemistry 173 (2015) 619–623

Table 3 Table 5
Figures of merit in the calibration curve obtained for hydroxyproline between 0.12 Comparison between acid hydrolysis using the AOAC method and alkaline hydrolysis
and 4.8 lg mL 1. using the proposed method. The results are expressed in mg collagen per g of tissue
analysed (mean of 5 replicates ± SD).
Equation Y = (0.2043 ± 0.0010)X + (0.0034 ± 0.0029)
1
1 Sample Collagen (mg g ) Paired t-test
LOD 0.03 lg mL
1
LOQ 0.11 lg mL Acid Alkaline texp tcrit
R2 0.9998
Sausage 13.76 ± 0.21 13.61 ± 0.22 1.11 2.31
Porcine Liver 8.27 ± 0.06 8.35 ± 0.15 1.09

Fig. 1. Efficiency profile of the hydrolysis of 383.8 lg collagen with the addition of
500 lL NaOH (2, 4, 8, 10 and 12.5 mol L 1), under the time and temperature
conditions established by Reddy and Enwemeka (1996) (20 min at 120 °C).
Recovery was calculated as the ratio between the determined and the expected
masses of hydroxyproline, assuming 12.5% (w/w) hydroxyproline in collagen (Kolar,
1990). Error bars represent the standard deviation (SD) of 4 replicates (n = 4) at each
point.
3.2. Study of alkaline hydrolysis
value is about ten times higher than the highest amount of colla-
gen analysed in frozen tissues by Reddy and Enwemeka (1996).
Figures of merit for the analytical curve used to quantify the
hydroxyproline after hydrolysis are shown in Table 3. Recovery
increased with increasing concentrations of NaOH until a maxi-
mum of approximately 95% was reached, after which the increase
of NaOH had no effect (Fig. 1). It is likely that under these condi-
tions, the hydrolysate still contains peptides to which hydroxypro-
line is bonded.
A NaOH concentration of 7 mol L 1 was established as a stan-
dard for subsequent assays. Higher concentrations (10 and
12.5 mol L 1) released enough heat to promote the boiling of the
hydrolysate during acidification.
The required reaction time to assure complete hydrolysis was
investigated. In the present study, the highest recoveries for the
amounts of collagen tested were generally obtained at and after
30 min; for the lowest amount (71.31 lg), 100% recovery was
achieved in 20 min, and no decrease was observed until 40 min
(Table 4). Given that the maximum recoveries for 378.18 lg colla-
gen were obtained within 40 min, this duration was used in subse-
quent assays. Hofman et al. (2011) chose to keep the concentration
of NaOH (2 mol L 1) constant and carried out hydrolysis for three
hours to ensure that the reaction was complete. However, a direct
comparison is not possible because the amounts of collagen hydro-
lysed by these authors were higher. Using between 50 and 75 lg of
collagen in the test tube, Huszar et al. (1980) found a 10% decrease
in recovery after 30 min, which was ascribed to the destruction of
hydroxyproline caused by a long exposure to NaOH. This effect was
not observed here.

The hydrolysis of collagen in an alkaline medium follows the
general hydrolysis mechanism for amides, where NaOH is con-
sumed during the reaction. Nucleophilic attack on the peptide car-
bonyl is rapid; breaking the C–N bond is slow. This reaction can be
accelerated by heat. The efficiency, therefore, depends on the
NaOH concentration and on the temperature and time of the reac-
tion (Chirita, 2000). Controlled conditions were adopted in this
work for the development of a reproducible method. Therefore,
the parameters involved in the hydrolysis reaction were studied
using a collagen standard. The temperature and time in the auto-
clave used for the initial conditions were the same as those
reported by Reddy and Enwemeka (1996). Volumes were chosen
for operational reasons; it was important to avoid the potential
leakage of samples in the autoclave because the tubes were not
sealed. The amount of collagen was kept constant (383.3 lg). This
3.3. Application of the method to real matrixes
The procedure was applied to porcine sausages and liver
acquired from the local market, and compared to acid hydrolysis
by the AOAC method. According to the AOAC method, 1:20 dilution
of the hydrolysate is the most usual procedure before spectropho-
tometric determination of hydroxyproline. To determine the best
dilution, pH after dilution and the addition of the buffer was veri-
fied for dilution factors of 1, 5, 10 and 20. Because a constant pH
value of 5.44 was obtained after the addition of the buffer for dilu-
tions of 1:10 and 1:20, a dilution factor of 10 was used here. Table 5
shows the results using the AOAC and the proposed methods. Stu-
dent’s paired t-test showed no significant difference (p > 0.05)
between the values obtained using acid and alkaline hydrolysis.
The methods are, therefore, equivalent with respect to the results

Table 4
Mean recovery (n = 4) ± standard deviation (SD) as a function of the duration of hydrolysis for 4 different amounts of collagen.

Amount of collagen (lg) Time (min)

10 20 30 40 50 60
71.37 0.93 ± 0.03 1.00 ± 0.02 1.03 ± 0.01 1.04 ± 0.02 – –
142.74 0.92 ± 0.01 0.98 ± 0.01 0.99 ± 0.02 1.02 ± 0.00 – –
285.47 0.90 ± 0.01 0.98 ± 0.01 1.00 ± 0.01 1.03 ± 0.02 – –
378.18 – – 0.98 ± 0.01 1.03 ± 0.01 1.03 ± 0.00 1.07 ± 0.01
 C.M.L. da Silva et al. / Food Chemistry 173 (2015) 619–623
obtained, but the reaction time was reduced from 16 h for the
AOAC method to 40 min for the proposed method.
4. Conclusion
Conditions were established for the reproducible alkaline
hydrolysis of collagen in fresh solid samples and in collagen solu-
tions and for the sensitive hydroxyproline assay of the hydroly-
sates. Small amounts of sample are required, i.e., 10- to 20-mg
samples that contain 3.5–19.1 mg g 1 collagen or a 200-lL solu-
tion containing 70–380 lg collagen. The method was tested with
sausages and liver, but in principle, different matrixes could be
used. Compared to acid hydrolysis by the AOAC method, the reac-
tion time was reduced from 16 h to 40 min. The non-collagen pro-
tein content, although it consumes the base during the process, did
not affect the yield because of the optimisation of the concentra-
tion of NaOH used. Compared to published collagen determination
by alkaline hydrolysis, this method substantially improved the
sensitivity of the determination (factor 5) by the hydroxyproline
assay. Only simple and basic instrumentation were necessary.
Acknowledgement
The authors thank the Brazilian agency ‘Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro’ (FAPERJ) for financial support.
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