determination of chondroitin sulfate content in raw materials

DIETARY SUPPLEMENTS

Determination of Chondroitin Sulfate Content in Raw Materialsand Dietary Supplements by High-Performance LiquidChromatography with Ultraviolet Detection After EnzymaticHydrolysis: Single-Laboratory Validation

DAVID JI

Analytical Laboratories in Anaheim, Inc., 2951 Saturn St, Unit C, Brea, CA 92821

MARK ROMAN

Tampa Bay Analytical Research, Inc., PO Box 931, Safety Harbor, FL 34695

JOSEPH ZHOU

NOW Foods Inc., 395 S. Glen Ellyn Rd, Bloomington, IL 60108

JANA HILDRETH

Blaze Science Industries, 4547 W. 171st St, Lawndale, CA 90260

A method to quantify chondroitin sulfate in raw

materials and dietary supplements at a range of

about 5 to 100% (w/w) chondroitin sulfate has been

developed and validated. The chondroitin sulfate is

first selectively hydrolyzed by chondroitinase ACII

enzyme to form un-, mono-, di-, and trisulfated

unsaturated disaccharides; the resulting

disaccharides are then quantified by ion-pairing

liquid chromatography with ultraviolet detection.

The amounts of the individual disaccharides are

summed to yield the total amount of chondroitin

sulfate in the material. Single-laboratory validation

has been performed to determine the repeatability,

accuracy, selectivity, limit of detection, limit of

quantification, ruggedness, and linearity of the

method. Repeatability precision for total

chondroitin sulfate content was between 1.60 and

4.72% relative standard deviation, with HorRat

values between 0.79 and 2.25. Chondroitin sulfate

recovery from raw material negative control was

between 101 and 102%, and recovery from finished

product negative control was between 105 and

106%.

Chondroitin sulfate (CS) is a negatively charged

polymeric glycosaminoglycan (GAG) consisting of

alternating glycuronic acid and N-acetylhexosamine

residues connected by �1-3 hexuronidic and

�1-4-N-acetylhexosaminidic bonds (1; Figure 1). It is closely

related to other GAGs such as dermatan sulfate, hyaluronic

acid, heparin, heparan sulfate, and keratan sulfate. CS

contains N-acetylgalactosamine (GalNAc) as the hexosamine

and glucuronic acid (GlcA) as the glycuronic acid moiety (2),

while other GAGs contain other hexosamine and/or

glycuronic acid residues. Either of the residues can be sulfated

at different positions.

CS is a major component of connective tissue and is

partially responsible for providing the flexibility of these

tissues. Oral administration of CS may help treat symptoms of

osteoarthritis (3–8), and, as a result, dietary supplements

containing CS that claim to promote healthy joints are readily

available. Predominant sources of CS raw materials in

commerce are bovine trachea, porcine skin and rib cartilage,

and shark cartilage.

Quantitative analysis of CS in CS raw materials and dietary

supplements containing CS raw materials has been extremely

challenging owing to the wide molecular weight variation of

CS polymers, its poor UV absorbance, and strongly ionic

nature. Other related GAGs may be present as impurities or

adulterants in CS materials, and, thus, any analytical

methodology designed to quantify CS must be selective for

CS in the presence of these other GAGs. Carbazole

reaction (9, 10), cetyl pyridinium chloride (CPC)

titration (11), and size exclusion chromatography (12) have

been used to characterize CS; however, these methods cannot

distinguish between CS and related GAGs, and are subject to

interferences in dietary supplement finished products. CPC

titration, in particular, has become a popular method for

determining CS purity; however, this method not only cannot

distinguish between CS and other GAGs, but it will give

positive results for any large moleculer anion, such as

carrageenan, proteins, and surfactants.

Enzymatic hydrolysis of CS followed by high-

performance liquid chromatography (LC; 1, 2, 13–22) has

been used to characterize CS raw materials and CS present in

tissues. The CS is treated with either chondroitinase ABC or

chondroitinase AC enzyme to selectively hydrolyze the

JI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 90, NO. 3, 2007 659

Received November 27, 2006. Accepted by AP March 8, 2007.Corresponding author's e-mail: [email protected]

polymer to resulting unsaturated disaccharide units (Figure 1).

Chondroitinase ABC will hydrolyze both CS and dermatan

sulfate (sometimes referred to as “chondroitin sulfate B”),

while chondroitinase AC is specific for CS. The resulting

unsaturated disaccharide units can then be separated and

quantified by ion-exchange chromatography (2, 13–19, 21, 22)

or reversed-phase chromatography (1, 17, 20), with either

ultraviolet (UV) detection (1, 2, 13–16, 18, 20), conductivity

detection (17), or precolumn derivatization with fluorescence

detection (19, 21, 22). None of these methods, however, has

been applied to dietary supplements, and none has been

validated rigorously.

With the prevalence of CS dietary supplements in the

marketplace, it is important to have an accurate and

reproducible analytical method for the quantitation of CS in

these products. An ion-pairing reversed-phase LC-UV

method utilizing enzymatic hydrolysis of CS was developed

and validated. The method uses aqueous extraction followed

by hydrolysis of the CS by chondroitinase AC II enzyme to

the unsaturated disaccharides. The unsaturated disaccharides

are then separated and quantified using gradient elution

ion-pairing LC with UV detection at 240 nm. The accuracy,

repeatability, linearity, range, selectivity, and ruggedness of

the method were demonstrated.

Experimental

Materials

CS raw materials from bovine trachea, porcine

skin/cartilage, and shark cartilage, and CS control material

from bovine trachea were obtained from Bioiberica

(Barcelona, Spain). Dietary supplement products containing

CS (hard-shell capsules, tablets, chewables, softgels, and

liquids) were obtained from commercial suppliers.

Descriptions of the dietary supplement products used in the

study are presented in Table 1.

Apparatus

(a) LC system.—Beckman 126 dual high pressure mixing

pumps (Beckman Coulter, Fullerton, CA), 168 diode array

UV detector, 507e autosampler, and 32 Karat software.

(b) Operating conditions.—Mobile phase flow rate,

1.1 mL/min; column temperature, ambient; injection volume,

30 �L; and detection, 240 nm.

(c) LC column.—Phenomenex Synergi Polar-RP, 4.6 �

150 mm, 4 �m particle size (Phenomenex, Torrance, CA).

(d) Analytical balance.—Accu-124 (Fisher Scientific,

Pittsburgh, PA), ±0.01 mg readability.

(e) Ultrasonic bath.—Model FS60H (Fisher Scientific).

(f) pH meter.—Model pH 500 (Oakton, Vernon Hills, IL),

±0.01 pH unit readability.

(g) Dry block heater.—Isotemp Dry Bath Incubator

(Fisher Scientific), maintained at 37�C.

(h) LC injection vials.—2 mL, with caps and

Teflon-coated septa.

(i) Limited volume inserts.—200 �L, for LC vials.

(j) Syringes.—25, 100, and 500 �L Luer-Lok.

Reagents

Note: Chemicals from other suppliers meeting the

specifications may also be used.

660 JI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 90, NO. 3, 2007

Figure 1. Structures of chrondroitin sulfate and unsaturated disaccharides resulting from enzyme hydrolysis bychondroitinase AC II enzyme.

(a) Solvents.—Acetonitrile, LC grade; water, LC grade;

hydrochloric acid, concentrated, ACS reagent grade.

(b) Tetrabutylammonium bisulfate.—Minimum 99.0%

(Fluka, St. Louis, MO; http://www.sigma-aldrich.com; Cat.

No. 86868).

(c) Tris-(hydroxymethyl)aminomethane (TRIS).—Sigma

(St. Louis, MO), Cat. No. T-1503.

(d) Sodium acetate.—Anhydrous (Sigma, Cat. No.

S-8750).

(e) Acetic acid.—Glacial (Sigma, Cat. No. A-0808).

(f) Sodium chloride.—ACS reagent grade.

(g) Bovine serum albumin.—1x Crystallized, �97%

(Sigma, Cat. No. A-4378).

(h) Chondroitinase AC II.—5 units (Seikagaku

America/Associates of Cape Cod, East Falmouth, MA, Cat.

No. 100335-1A, http://www.acciusa.com).

(i) Mobile phase A.—Weigh 340 mg tetrabutylammonium

bisulfate and transfer into a 1000 mL volumetric flask.

Dissolve and dilute to volume with water. Degas.

(j) Mobile phase B.—Weigh 340 mg tetrabutylammonium

bisulfate and dissolve in 330 mL deionized (DI) water. Add

acetonitrile to 1000 mL. Sonicate and filter.

(k) 0.12 M HCl.—Carefully add 1 mL concentrated HCl

to 99 mL water and mix well.

(l) 6 M HCl.—Carefully add 50 mL concentrated HCl to

50 mL water and mix well.

(m) TRIS buffer solution.—Dissolve 3 g TRIS, 2.4 g

sodium acetate, 1.46 g sodium chloride, and 50 mg crystalline

bovine serum albumin in 100 mL of 0.12 M HCl. Adjust pH to

7.3 with 6 M HCl.

(n) Enzyme solution.—Dissolve 5 units of chondroitinase

AC II enzyme in 0.5 mL water. Store at <0�C when not in use.

(o) Dilution solution.—Prepare at least 20 mL of a

solution containing 80% mobile phase A and 20% mobile

phase B.

(p) Reference standards.—See Table 2. Purities were

obtained from the supplier’s certificate of analysis. These

purities were determined by chromatographic purity, water

content, and residual solvent content. No independent

confirmation of the purity was performed.

Preparation of Test Solutions

(a) Preparation of standard solutions.—Accurately

weigh about 2 mg �Di-0S and 10 mg each of �Di-4S and

�Di-6S (Table 2), and transfer into a 50 mL volumetric flask.

Dissolve and dilute to volume with water. This is the stock

instrument calibration solution.

(b) Instrument calibration solutions.—Prepare serial

dilutions of the stock instrument calibration solution in water


Table 1. Materials

Material No. Type Composition Claim

1 Raw material CS from bovine trachea Pure

2 Capsules CS 250 mg CS/capsule

3 Chewables CS + glucosamine, ascorbic acid 125 mg CS/wafer

4 Tablets CS + glucosamine sulfate (from marine source) 400 mg CS/tablet

5 Softgels CS + glucosamine 200 mg CS/softgel

6 Raw material CS from porcine Pure

7 Raw material CS from shark cartilage Pure

8 Tablets CS + ascorbic acid, manganese, herbs 200 mg CS/tablet

9 Liquid CS + glucosamine, MSM, ascorbic acid, manganese 800 mg CS/29.57 mL

Table 2. Reference standards

Name Abbreviation Supplier

2-Acetamido-2-deoxy-3-O-(�-D-gluco-4-enepyranosyluronic acid)-D-galactose �Di-0S Sigma

2-Acetamido-2-deoxy-3-O(�-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose �Di-4S Sigma

2-Acetamido-2-deoxy-3-O-(�-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose �Di-6S Sigma

2-Acetamido-2-deoxy-3-O-(2-O-sulfo-�-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose �Di-di(2,6)S ICNa

2-Acetamido-2-deoxy-3-O-(�-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose �Di-di(4,6)S ICN

2-Acetamido-2-deoxy-3-O-(2-O-sulfo-�-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose �Di-tri(2,4,6)S ICN

a Now MP Biomedicals, Solon, OH.

at concentrations of about 2, 8, 20, 40, and 100 �g/mL �Di-4S

and �Di-6S, and 0.4, 1.6, 4, 8, and 20 �g/mL �Di-0S. A

possible dilution scheme is shown in Table 3.

(c) CS control solution.—Accurately weigh about 100 mg

CS control sample into a 50 mL volumetric flask. Add about

30 mL water, and sonicate until the sample is completely

dissolved (about 15 min). Dilute to volume with water and

mix well. Label Control Solution 1.

(d) Sample test solutions.—(1) Raw materials.—

Accurately weigh about 200 mg CS raw material into a

100 mL volumetric flask. Add 60 mL water, and sonicate until

the sample is completely dissolved (about 15 min). Dilute to

volume with water and mix well. Label Test Solution 1.

(2) Tablets.—Determine the average tablet weight by

weighing 20 tablets and calculating the average weight of

1 tablet. Grind the 20 tablets to a powder and mix. Accurately

weigh a test portion containing the equivalent of about 200 mg

CS into a 100 mL volumetric flask. Add about 60 mL water

and sonicate for 15 min. Dilute to volume with water and mix

thoroughly. Filter ca 1–2 mL Test Solution 1 through a 0.2 �m

PTFE syringe filter. Label Test Solution 1.

(3) Capsules.—Determine the average capsule content weight

by weighing 20 capsules. Record the weight. Empty and

combine the capsule contents. Thoroughly clean the capsule

shells using a swab and/or compressed air. Weigh and record

the weight of the empty capsule shells:

Average capsule fill weight, g =C S�

20

where C = total weight of 20 capsules and S = total weight of

20 capsule shells. Proceed as directed in section (d)(2) for

tablets. (4) Liquid formulations.—Thoroughly mix the

sample. Accurately weigh an amount of test portion

containing the equivalent of about 200 mg into a 100 mL

volumetric flask. Dissolve in and dilute to volume with water.

Mix thoroughly. Label Test Solution 1.

(e) Enzymatic hydrolysis of control solution and test

solution.—Pipet 20 �L TRIS buffer solution, 30 �L enzyme

solution, and 20 �L Control Solution 1 or Test Solution 1 into

a 2 mL LC injection vial with a 200 �L insert. Place the vial in

a 37�C dry bath or water bath for 3 h. Allow to cool room

temperature. Using an automatic pipettor or gas-tight syringe,

carefully transfer the solution into an LC vial. Rinse the

200 �L insert with exactly 100 �L mobile phase A using a

calibrated automatic pipettor or gas-tight syringe, and

quantitatively transfer this into the LC vial. Dilute to 1.00 mL

by adding 830 �L mobile phase A to the LC vial. Mix well.

Label Control Solution 2 or Test Solution 2 – Treated.

Determination

(a) Mobile phase gradient program.—Elute the analytes

with the linear gradient program of mobile phases A and B

shown in Table 4.

(b) System suitability tests.—Equilibrate the LC system

with the mobile phases for at least 30 min until a stable

baseline is obtained. Inject each of the 5 instrument calibration

solutions. Use linear regression to determine the slopes,

y-intercepts, and correlation coefficient (r2) of the calibration

lines for �Di-0S, �Di-4S, and �Di-6S. The correlation

coefficient of the calibration line for each component must be

>0.998 (for Di-0S >0.995). The tailing factor for all the

components in the linearity standards must be between 0.80

and 1.5. Inject Control Solution 2 – Treated and calculate the

total amount of CS in the control material [Calculations

(a)–(h)]. The recovery should be within ±3% of the

specification.

(c) Injection.—Make single injections of each standard

and test solution. After every 20 sample injections, and after

all of the sample injections are completed, make a single

injection of each standard solution.

(d) Retention times.—The approximate retention times

for each analyte are presented in Figure 2.

(e) Chromatograms.—Representative standard and

sample chromatograms are presented in Figures 2–4.

Calculations

(a) The amount of �Di-0S in �g/g, representing

unsulfated CS in the sample, is calculated as follows:

P b

m

V

WD0 0

0

��

where P0 = peak area of �Di-0S in sample chromatogram; b0 =

y-intercept of calibration curve for disaccharide �Di-0S; m0 =

slope of calibration curve for disaccharide �Di-0S; V =


Table 3. Preparation of instrument calibration

solutions

Calibrationsolution

Volume of stockpipetted, mL

Final volume(flask size), mL

1 25 50

2 10 50

3 5 50

4 2 50

5 1 100

Table 4. Linear mobile phase gradienta

Time, min Mobile Phase A, % Mobile Phase B, %

0 80 20

0–7.0 35 65

7.0–12.0 35 65

12–12.5 80 20

a The column should be re-equilibrated at the starting mobile phaseconditions for at least 10 min after each injection.

volume of Test Solution 1 = 100 mL; W = sample weight, in g;

and D = dilution factor = 50.

(b) The amount of �Di-4S in �g/g, representing CSA in

the sample, is calculated as follows:

P b

m

V

WD4 4

4

��






(c) The amount of �Di-6S in �g/g, representing CSC in

the sample, is calculated as follows:

P b

m

V

WD6 6

6

��






(d) The amount of �Di-di(2,6)S in �g/g in the sample is

calculated as follows:

P b

m

V

WD F

2 6 6

6

,�

� � �

where P2,6 = peak area of �Di-di(2,6)S in sample

chromatogram; b6 = y-intercept of calibration curve for

disaccharide �Di-6S; m6 = slope of calibration curve for

disaccharide �Di-6S; V = volume of Test Solution 1 =

100 mL; W = sample weight, in g; D = dilution factor = 50;

and F = molecular weight conversion between �Di-6S and

�Di-di(2,6)S = 1.190.

(e) The amount of �Di-di(4,6)S in �g/g in the sample is


P b

m

V

WD F

4 6 6

6

,�

� � �

where P2,6 = peak area of �Di-di(4,6)S in sample






�Di-di(4,6)S = 1.190.

(f) The amount of �Di-tri(2,4,6)S in �g/g in the sample is


P b

m

V

WD F

2 4 6 6

6

, ,�

� � �

where P2,4,6 = peak area of �Di-tri(2,4,6)S in sample






�Di-tri(2,4,6)S = 1.380.

(g) The total amount of CS in �g/g in the sample is the sum

of �Di-0S, �Di-4S, �Di-6S, �Di-di(2,6)S, �Di-di(4,6)S, and

�Di-tri(2,4,6)S.

(h) % (w/w) is calculated from �g/g as follows:

% (w/w) =�g g/

10000

(i) Milligrams per tablet (mg/tab) is calculated from �g/g

as follows:

�g gTW

/

1000�

where TW = the average tablet weight in grams.


Figure 2. Stock standard solution chromatogram.Peak assignments and approximate retention times:

(1) �Di-0S (2.5 min), (2) �Di-6S (5.9 min), (3) �Di-4S (6.3).

Not shown: �Di-di(2,4)S (9.6 min), �Di-di(2.6)S (9.9 min),

�Di-tri(2,4,6)S (12.0 min).

Figure 3. CS raw material from bovine trachea sample

chromatogram. Peak assignments: (1) �Di-0S,

(2) �Di-6S, (3) �Di-4S.

(j) Milligrams per capsule (mg/cap) is calculated from

�g/g as follows:

�g gFW

/

1000�

where FW = the average capsule fill weight in grams.

(k) Milligrams per milliliter (mg/mL) is calculated from

�g/g for liquid samples as follows:

�g gSG

/

1000�

where SG = the specific gravity of the sample in g/mL.

Validation Design

(a) Linearity.—The 5 instrument calibration solutions

were injected at the beginning of each chromatographic

injection sequence, after every 20 sample injections, and at the

end of each sequence. A 5 point standard curve was generated

for all 3 analytes, and the slope, y-intercept, correlation

coefficient, and relative standard deviation (RSD) of the

standard curve were calculated for using the average peak

areas at each calibration point on each day.

(b) Repeatability.—Four replicates of each of the

Materials 1–5 (Table 1) representing a CS raw material, a

hard-shell capsule product containing CS, a tablet product

containing CS, a chewable product containing CS, and a

liquid product containing CS were prepared on each of 3 days,

for a total of 12 replicate preparations of each material. The

within-day, between-day, and total repeatability of the total

CS content were calculated. The HorRat value (23) for each

material was also calculated. In addition, 4 replicates of each

of the Materials 6–9 were prepared on a single day to

demonstrate the applicability of the method to these materials.

The within-day repeatability was calculated for these

materials.

(c) Accuracy.—(1) CS raw material.—Heparin, a related

GAG, was used as a negative control. About 200 mg heparin

was transferred into ten 100 mL volumetric flasks; 300 mg

bovine trachea CS raw material used in the repeatability study

(Material 1 in Table 1) was added to 3 of the flasks, 200 mg of

the same CS raw material was added to another 3 of the flasks,

100 mg of the CS raw material was added to another 3 of the

flasks, and the 10th flask was used as a negative control. Each

of the spiked negative controls was prepared and analyzed

according to the method on 3 separate days. (2) Spike recovery

of dietary supplement finished products.—A dietary

supplement tablet product containing glucosamine HCl and

methyl sulfonylmethane (MSM) was used as a negative

control for spike recovery study of dietary supplement

finished products. The tablets were first ground to a powder

and homogenized. About 500 mg of tablet negative control

material was transferred into ten 100 mL volumetric flasks.

The tablet negative control was then spiked with the bovine

trachea CS raw material used in the repeatability study using

the same procedure as described for the CS raw material spike

recovery study. Each of the spiked negative controls was

prepared and analyzed according to the method on 3 separate

days.

(d) Ruggedness.—A Youden ruggedness study was

conducted on the bovine trachea raw material, varying the

7 factors presented in Table 5 (24).


Figure 4. CS raw material from shark cartilage sample

chromatogram. Peak assignments: (1) �Di-0S, (2) �Di-6S,

(3) �Di-4S, (4) �di-di(2,6)S, (5) �di-di(4,6)S.

Table 5. Youden ruggedness testing

Parameter High value Low value Factor

Sonication time, min A = 30 a = 15 0.45

Sample weight, mg CS B = 200 b = 100 –0.45

Digestion temperature, �C C = 42 c = 37 –0.8

Concentration of enzyme solution D = 5 units/0.5 mL d = 5 units/1.0 mL –0.05

pH of TRIS buffer solution E = 7.1 e = 7.5 0.8

Injection volume, �L F = 50 f = 30 1.3

Detector wavelength, nm G = 240 g = 235 –0.6

(e) Selectivity.—The selectivity of the method was

demonstrated by injecting solutions of non-CS ingredients

typically found in CS-containing dietary supplements,

including glucosamine, MSM, vitamins, and minerals, into

the chromatographic system after treatment with enzyme. In

addition, possible contaminants and/or adulterants, such as

carrageenan, dermatan sulfate, and heparin, were subjected to

the same sample preparation procedure and injected into the

chromatographic system. The potential chromatographic

interference of hyaluronic acid (HA) was also investigated.

(f) Stability.—The stabilities of the chondroitinase AC II

enzyme in solution and the sample solution were evaluated

over the course of the study. (1) Enzyme stability.—A portion

of the enzyme solution used to prepare the precision samples

from Day 1 was stored at –20�C. A sample of the bovine

trachea CS was prepared and tested after 1 week using this

enzyme solution after warming to room temperature. The

result from this experiment was compared to the average

result obtained in the repeatability study for this material. (2)

Sample solution stability.—A portion of one of the digested

bovine CS sample solutions prepared on Day 1 of the

precision study was retained and injected on Day 2 of the

precision study. The result from this experiment was

compared to the average results obtained in the precision

study for this material.

Results and Discussion

The total CS content is calculated as the sum of the

individual disaccharides generated from the enzymatic

hydrolysis using chondroitinase AC II enzyme. Because of

the high price and limited quantities of the di- and trisulfated

reference standards, these components were quantified

against the monosulfated reference standards using a

molecular weight correction factor, as the molecules should

have identical molar absorptivities at 240 nm.

Selectivity

The selectivity of the method was demonstrated by

preparing mixtures of CS in combination with other

ingredients found in typical dietary supplements that could

interfere with the assay. These ingredients included the

divalent minerals calcium sulfate, magnesium chloride, zinc

chloride, and cupric sulfate, as it is believed that some divalent

metals can cause deactivation of the chondroitinase enzyme.

Other ingredients included in the selectivity study included

glucosamine HCl, MSM, chromium(III) chloride, dermatan

sulfate, carrageenan, and HA.

Table 6 presents the mixtures used in the selectivity study.

The percent recovery of CS from these mixtures percent was

calculated. No observable interference was found for any of

the ingredients with the exception of HA. Chondroitinase AC

II cleaves HA to the disaccharide unit �Di-0SHA, a

stereoisomer of the �Di-0S produced from CS that cannot be

resolved with the chromatographic method. Because

�Di-0SHA is the only disaccharide produced from HA, its

presence in samples can generally be detected by a very high

ratio of �Di-0S to the other disaccarides. Because HA is

considerably more expensive than CS, it is not considered a

source of economic adulteration.

Linearity

A 5-point calibration curve covering approximately

2 orders of magnitude in concentration range was generated

for each day of analysis. Linear regression was used to

calculate the slope and y-intercept of the standard curve for

each disaccharide. The correlation coefficient and residuals of

each standard curve for each day was determined. The data

showed that standard curves were linear from a concentration

of about 0.2 to about 10 �g/mL for �Di-0S, from about 1.4 to

70 �g/mL for �Di-4S, and from about 2 to 100 �g/mL for

�Di-6S. Table 7 summarizes the linearity data. Figure 5


Table 6. Selectivity matrixes

CS added, mga

Other ingredients mg CS found Recovery, %

288.7 Calcium sulfate 244.8 281 97.3

Magnesium chloride 523.2

Zinc chloride 289.0

Cupric sulfate 55.2

Glucosamine HCl 407.0

MSM 244.8

226.3 Chromium(III) chloride 79.8 226 100

152.4 Dermatan sulfate 45.5 155 102

152.4 Carrageenan 40.5 152 99.7

0.0 Hyaluronic acid 33.4 25.3b

NAc

a Weights corrected for purity.b Calculated as �Di-0S.c NA = Not applicable.

presents a typical residual plot for �Di-6S, with residuals

expressed as a percent. The residual plot does not show any

trend; the largest residual is at the lowest concentration, which

is near the limit of quantification for the method.

Accuracy

CS raw material.—Heparin was used as a negative control

for the raw material spike recovery study due to its similar

chemical structure compared to CS and its availability. CS

was spiked into heparin at levels corresponding to about 50,

100, and 200% of the heparin weight, or 33, 50, and 60% of

the total CS + heparin weight. These amounts also

corresponded to 50, 100, and 200% of the amount of CS that

would be weighed in a typical sample preparation. The

amount of each disaccharide and total amount of CS was

calculated. The spike recovery study was repeated on 2

additional days to obtain repeatability data. Results are

presented in Table 8. The recovery was calculated based upon

the repeatability study results for the bovine trachea CS raw

material of 92.36%. Average recoveries ranged from 100.8 to

101.6%, and total RSDs from 0.98 to 2.84%, indicating

excellent recovery and repeatability for the raw material.

Although f-tests indicated differences in the means between

days for the recovery study, this was primarily due to the

exceedingly tight within-day results.

Dietary supplements.—Spike recovery studies were used

to determine the recovery of CS from a complex dietary

supplement matrix. A commercial tablet product containing

both glucosamine HCl and methyl sulfonylmethane, both

commonly found in supplements containing CS, was selected

as a negative control (matrix blank). CS was spiked into the

powdered tablet material at levels corresponding to 20, 40,

and 60% of the negative control weight, or 16.7, 28.6, and

37.5% of the total weight. These values correspond to 50, 100,

and 150% of the amount of CS that would be found in a

typical sample preparation. The amount of each disaccharide

and total amount of CS was calculated. The spike recovery

study was repeated on 2 additional days to obtain repeatability

data. Table 9 summarizes the accuracy results for the dietary

supplement spike recovery study. Average recoveries of CS

were approximately 105–106% for all 3 levels, with RSDs

ranging from 2.0–3.5%.

Repeatability

Within-day, between-day, and total standard deviations

were calculated for each of the disaccharides, as well as the

total CS content using single-factor analysis of variance

(ANOVA) with a significance level (-value) of 0.5

(95% confidence interval). The method exhibited very good

repeatability for total CS in each material. Repeatability RSDs

ranged from 1.60% for the bovina trachea CS raw material to

4.72% for the multicomponent hard-shell capsule finished

product. The Horwitz ratio (HorRat) can be a useful index of

method performance with respect to precision, and is a ratio of

the observed RSD to the predicted RSD (23, 25). The

predicted RSD is calculated as 2C–0.15

, where C is the mean

concentration of the analyte in the matrix. Originally

developed using between-laboratory RSD (RSDR), the

HorRat has recently been applied to single-laboratory

validations (SLV; RSDr; 25). Although HorRat values are not

considered applicable to enzymatic methods or polymeric

materials (23), they were calculated for comparison with

traditional methods for small molecule species. HorRat values

ranged from 0.79 to 2.25. Table 10 summarizes the


Table 7. Linearity data

Slopea y-Interceptb rc RSDd, %

�Di-0S 17087 424 0.99997 1.3

�Di-4S 15106 –12931 0.9993 2.5

�Di-6S 14507 –9912 0.9998 1.1

a Units in mAU*s*mL/�g.b Units in mAU*s.c Coefficient of determination.d % RSD is calculated from the square root of the average variance of the calibration curves. The variance of the calibration curve is calculated

as:1 2

1N mWi Yi F Xi

i

N

��

� ( ( )) where N = number of calibration points (5), m = number of coefficients to determine (2), Wi = weight factor of

the calibration point (1 for all points), Yi = Y-value (peak area) of the calibration point number “i”, Xi = X-value (concentration) of thecalibration point number “i”, and F(x) = regression equation.

Figure 5. �Di-6S linearity residual plot.

repeatability results for total CS in each material. Results are

the average of all 12 sample preparations.

Single-day repeatability studies were conducted on

Materials 6–9 (Table 1) to demonstrate applicability of the

method to these materials. All materials showed excellent

within-day repeatability, consistent with the results found with

the other material (Table 11). The shark cartilage CS was the

only raw material found to contain the di- and trisulfated

disaccharides. These accounted for approximately 20% of the

total CS. Of the finished products tested, only Material 4

(Table 1) was found to contain di- and trisulfated

disaccharides, indicating that this material may contain

trisulfated from shark cartilage. No CS was found in the liquid

dietary supplement (Material 9 in Table 1). This material was

then spiked with bovine trachea CS raw material at the

expected concentration, with excellent recovery (Table 12).

This limited spike recovery study on this material indicates

that the product did not, in fact, contain any measurable CS,

and that the method is applicable to liquid supplements.

Ruggedness

A Youden ruggedness trial was conducted on 7 method

variables that could affect the results of the analysis. Eight

experiments were conducted by varying each of the

7 variables in specific high/low combinations (24). The

variables included sample sonication time, sample weight,


Table 8. CS raw material recoverya

Level 1 Level 2 Level 3

Nominal amount of heparin added, mg 200 200 200

Nominal amount of CS added, mg 100 200 300

Nominal CS concentration, % 33 50 60

Recovery, % 101.6 101.1 100.8

RSD, % 2.8 1.8 0.98

a Average results for 3 replicates on 3 days.

Table 9. Finished product recoverya

Level 1 Level 2 Level 3

Nominal amount of negative control added, mgb

500 500 500

Nominal amount of CS added, mg 100 200 300

Nominal CS concentration, % 16.7 28.6 37.5

Recovery, % 105.6 105.4 105.8

RSD, % 2.0 3.5 3.1

a Average results for 3 replicates on 3 days.b Negative control consisted of glucosamine 175, glucosamine sulfate 175, MSM 82, ascorbic acid 6, malic acid 3, citric acid 4, fructose 20,

potassium sorbet 1, sodium benzoate 1, magnesium stearate 0.5, stearic acid 1, cellulose 230, rice maltodextrin 300, lemon granules 0.3,and silica 0.2 (all units g/kg).

Table 10. Repeatability results

Materiala

nb Resultc Within-day SDc Between-day SDc Total SDc Total RSD, % PRSDd HorRat

A 12 923.6 8.31 12.2 14.8 1.60 2.02 0.79

B 12 740.4 5.55 34.5 34.9 4.72 2.09 2.25

C 12 21.70 0.340 0.943 1.00 4.62 3.55 1.30

D 12 202.6 2.22 8.92 9.2 4.54 2.54 1.79

E 12 210.9 3.69 8.67 9.4 4.47 2.53 1.77

a A = CS raw material from bovine trachea, B = hard-shell capsules, C = chewables, D = CS + glucosamine tablets, and E = CS + glucosaminesoftgels.

b n = Total number of samples tested.c Results in mg/g.d PRSD = Predicted RSD, calculated as 2C–0.15 (see refs. 23 and 24).

enzyme hydrolysis time, enzyme concentration, enzyme

buffer pH, injection volume, and detector wavelength. The

Youden ruggedness trial yields unitless values that give an

indication of the relative effects of each factor on the results.

Outliers in these results indicate which factors affect the

method results, and thus must be controlled or monitored. The

results of the Youden ruggedness trial (Table 5) show that

none of the factors examined is critical to the results within the

ranges examined, indicating a very rugged method. The

overall RSD for all 8 experiments was 1.1%.

Stability

Enzyme solution stability was demonstrated over the

course of 1 week. A sample of the bovine trachea CS raw

material was treated with enzyme solution that had been

stored at –20�C for 1 week. Avalue of 91.0% CS was obtained

for this sample, compared with an average result of 92.35%

obtained during the precision study using freshly prepared

enzyme, for a 98.5% recovery.

One of the bovine trachea CS raw material sample

preparations from the repeatability study was retested after

24 h to demonstrate sample solution stability. The recovery

was 99.8% after 24 h, indicating that the hydrolyzed sample

solution can be stored for 24 h at room temperature before

analysis.

Disaccharide Profiles

The average results for the disaccharides formed from the

enzymatic hydrolysis of CS for the 3 raw materials used in the

validation study (bovine trachea, porcine, and shark cartilage)

are presented in Table 13. Each material yields a distinct

disaccharide profile similar to what Karamanos et al.

found (18), however differences were also observed. No

disulfated disaccharides were found in the bovine trachea CS

raw material, for example, while Karamanos et al. found

approximately 10% disulfated disaccharides. Although it

appears that the disaccharide profile may aid in identification

of the source of CS raw materials, at present there is

insufficient data to establish representative ratios for each of

the materials.


Table 11. Within-day repeatability results

Materiala

nb Resultc SDc RSD, %

F 4 887 2.2 0.25

G 4 842 9.2 1.1

H 4 176.2 3.14 1.8

I 4 NDd

NAe

NA

a F = CS raw material from porcine source, G = CS raw material from shark cartilage, H = CS tablets, and I = CS + glucosamine + MSM liquidsupplement.

b n = Total number of samples tested.c Results in mg/g, SD = standard deviation.d ND = Not determined.e NA = Not applicable.

Table 12. Spike recovery results from liquid dietary

supplement

Amount CSadded, mg

Amount CSrecovered, mg Recovery, %

838 835 99.6

Table 13. Disaccharide profilesa

Bovine trachea Porcine Shark cartilage

�Di-0S 6.67 (2.06) 5.28 (0.176) 2.92 (0.081)

�Di-4S 53.8 (0.91) 62.9 (0.115) 28.2 (0.275)

�Di-6S 31.9 (0.66) 20.5 (0.200) 38.9 (1.11)

�Di-di(2,6)S NDb

ND 1.04 (0.084)

�Di-di(4,6)S ND ND 13.2 (0.163)

�Di-tri(2,4,6)S ND ND ND

a Results in % (w/w); standard deviation in parentheses.b ND = Not determined.

Conclusions

An enzymatic LC-UV method was developed and an SLV

study performed for the determination of CS in raw materials

and dietary supplements. The method was shown to be

accurate, repeatable, and rugged for the determination of CS

in these materials, and it does not suffer from interferences

that affect results of other methods for the analysis of CS

materials. The disaccharide profile resulting from the

enzymatic hydrolysis of CS shows potential for identifying

the source of the CS material. Based upon the SLV results, this

method is ready for full collaborative study.

Acknowledgments

This project was funded by a contract with the National

Institutes of Health, Office of Dietary Supplements, Bethesda,

MD.

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determination of chondroitin sulfate content in raw materials

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