STUDY DESIGNFOR Cr-PICOLINATE (200Μg)

The appropriate study design is given as follows –

 1 x 1 Design,
 One Healthy subject (Reference) and one patient
(Test).
 Single dose.

Volunteers
Number of Volunteers: Two (One healthy and one
patient)

Inclusion Criteria (Healthy Volunteers)

1. Adult healthy male of 25years.

2. Height of 5feet 7inch.
3. Weight of 60kg±5kg.

a) Abnormality on clinical examination :

No.
b) History of illness in the past 8 weeks :
No.

Exclusion Criteria
a) Consumption of tobacco in any form :
No.
b) Addiction to alcohol or history of any drug abuse
: No.
c) History old kidney or liver dysfunction :
No.
e) History of drug allergy to the test drug or any drug
Chemically similar to the drug under investigation :
No.
f) Administration/intake of any prescription or OTC :
No.
medication for 2weeks before the study.
g) Patients suffering form any chronic illness such :
No.
as arthritis, asthma, etc.
h) Subjects suffering form any psychiatric (acute or
: No.
chronic) illness.
i) Participation in any bioavailability/bioequivalence
study in the past 12 weeks. :
No.
j) intake of barbiturates or any enzyme-inducing drug
in the past 3 months. :
No.
k) HIV – positive volunteers. :
No.
Conduct of the study

a) Time of administration of the medication (test and

reference) was in the morning and the same time
strictly adhered to during entire study.
b) Tea, coffee and caffeine/xanthine-containing
beverages and foods was not allowed during each
phase of the study.
c) The volunteer fasted for 12 hours predosing and will
have a food just before the dose. (Break fast).
d) Uniform and identical meals will be provided an
identical time to all volunteers from the time they are
admitted and till the time they are housed.
e) A precise work schedule will be prepared in advance
and explained to the volunteers.
 f) The exact time of drug administration, sample
collection and meals was strictly adhered to and the
actual time of each activity is properly and accurately
documented.

Sl.No Time (hrs) Sample No. Time

Intervals
(hrs)
1. 8.20 AM S0 0.0
2. 8.50 AM S0.5 0.5
3. 9.50 AM S1 1.0
4. 10.50AM S2.0 2.0
5. 11.20AM S2.5 2.5
6. 11.50 AM S3.0 3.0
7. 12.20 PM S3.5 3.5
8. 12.50 PM S4.0 4.0
9. 1.50 PM S5.0 5.0
10. 2.50 PM S6.0 6.0
11. 4.50 PM S8.0 8.0
12. 6.50 PM S10.0 10.0
13. 8.50 PM S12.0 12.0
14. 12.50 AM S16.0 16.0
15. 8.50 AM S24.0 24.0

Sl.N Meals Time Ingredients and Amounts.

o
1. Morning 08.15 5slice of bread, 50gm of Amul
Breakfa AM. butter, 2 goodday biscuits and
st 250ml of water.
2. Tablet 8.50 With 200ml of water.
(Sample AM.
Cr-
picolinat
e)
200mcg
.
3. Lunch 2.10 5puris, 100gm rice, curry, 3type of
PM. vegetables, 1 papad, onion salad,
400ml of water.
4. Dinner 9.00 3 chappatis, Paneer, vegetables,
 PM. Rice 100gm, and water 400ml.

g) Test tubes/vials required for sample collection was

labeled in advance and kept ready.
h) Standard quantity of water (one glass: 200ml) will be
allowed for proper ingestion of the supplement.
i) The investigator provided the medication to the
volunteer and the volunteer consumed the dose in
the presence of the investigator.
j) After ingestion of the medication, the volunteer was
not allowed to resist in supine position at least for
2hours to ensure no gastric emptying, subsequently,
although the volunteers are ambulatory, any
strenuous physical or mental activity will not be
permitted.
k) Blood samples will be collected using disposable
needles, syringes etc.
l) Using disposables syringes multiple blood sample
collection was collected.
m) Care was taken not to withdraw more than
250ml of blood per volunteer in one analysis.
n) An attending physician was present at all times to
oversee the conduct of the investigation as well as to
answer any queries by the volunteer.
o) An emergency medical equipment and support
service was available and accessible throughout the
investigation.

Blood sampling
a) Adequate number (at least three) of venous blood
samples was collected during the absorption phase
and was spread over the expected duration of the
absorption phase.
b) At least 3 blood samples were collected around the
expected time of peak blood levels. This can be
made possible by adjusting 1 to 2 points each from
absorption points (last ones) and distribution points
(earlier ones) as near the Cmax as possible.
c) Sampling containers was sterilized using dry heat.
While transferring the sample from the syringe the
needle is removed and the syringe was slowly
emptied along the sides of the container.
d) The sampling container contained an adequate
amount of anticoagulant. The anticoagulant (in case
of plasma) was chemically compactable with the
dose and did not increase the volume of blood
sample stored in the container.
e) The separation of the plasma or serum was done
immediately following the collection stored in two
identical containers with identical labels.
f) Once separated, the plasma, serum or the biological
fluids collected, was immediately frozen to –100C to –
200C till the time of analysis.
Analytical Methods

Column – 250 x 4.6mm 5µ C18 Waters.

Mobile phase - 55% MeOH(v/v), 45%H20(v/v), 3mMTBAHS,
1 x 10-4
5Br-8HQ and 20mM acetate buffer(pH-
7.7).

Chromatographic conditions –
Wavelength – 260nm.
Flow rate – 1.0ml/min.
Injection volume - 100µl.

Standard preparation –
Pipette out 0.1ml of 0.0004M5Br-8HQ in methanol in
a test tube, evaporate and add 0.1ml Prepared 50ppm Cr
(II) stock solution. Add 1ml plasma and + 0.2ml of acetate
buffer + 1ml of chloroform. Vortex the mixture for 2min,
centrifuge and evaporate the supernatant layer, add 0.1ml
of mobile phase and inject 100µl of the aliquot in to the
chromatograph.

Sample preparation –
 0.5ml of plasma was taken in a test tube followed by
the addition of 0.1ml of 0.0004M5Br-8HQ in methanol +
0.2ml of acetate buffer + 1ml of chloroform. Vortex the
mixture for 2min, centrifuge and evaporate the
supernatant layer, add 0.1ml of mobile phase and inject
100µl of the aliquot in to the chromatograph.

Evaluation Parameters –

Validation data for Chromium picolinate for bioavailability

Parameter
Method Chelate formation with 5Br-
8HQ and determination by
HPLC.
Ion-pair reagent TBAHS.
Sensitivity/LOQ 0.1µg/ml.
Linearity (standar curve 0.1-19.77 µg/ml.
samples)
Quality control samples HLOQ – 0.299µg/ml.
MLOQ- 7.9633µg/ml.
LLOQ – 15.428µg/ml.
Precision of standard 8.7% @ 0.10 µg/ml.
curve(%CV) 4.2% @ 19.77 µg/ml.

Precision of QC samples. 8.8% @ 0.10 µg/ml.

Accuracy of standards.(%)
Accuracy of QC samples (%)
3.5% @ 15.428 µg/ml.
99.3% @ 0.10 µg/ml.
99.5% @ 19.77 µg/ml.
100.3% @ 0.299 µg/ml.
101% @ 15.428µg/ml.
Stability
Freeze-thaw 2 cycles.
Processed sample stability 16.00hrs @ 26OC.
at RT
Long term at –200C 72 hours.
Recovery
Low 100.3% @ 0.3 µg/ml.
Med 90.2% @ 8.01µg/ml.
High 90.4% @ 15.501 µg/ml.

Results
Mean chromium plasma levels (µg/ml) for the test and
reference subjects. Values are mean ± SD.
TEST REFERENCE

HOUR0.0 0.00 0.00 0.00 0.00

HOUR 0.5 10.25 0.36 0.33 0.41
HOUR 1.0 12.41 11.76 13.34 12.51
HOUR 2.0 15.49 12.71 16.59 13.57
HOUR 2.5 17.93 13.50 18.93 14.06
HOUR 3.0 19.17 13.44 10.16 13.90
HOUR 3.5 10.08 12.95 10.68 13.56
HOUR 4.0 10.10 13.02 10.64 12.54
HOUR 5.0 10.11 12.83 10.42 12.38
HOUR 6.0 18.63 11.71 18.71 12.09
HOUR 8.0 17.20 11.92 16.44 11.84
HOUR 15.62 12.07 14.79 11.56
10.0
HOUR 14.27 11.60 13.66 11.41
12.0
HOUR 11.29 10.64 11.06 10.47
16.0
HOUR 10.42 10.23 10.37 10.15
24.0

Mean test and reference parameters for chromium chelate

are mean ± SD.
TEST REFERENCE

Cmax 11.64 12.46 12.05 13.11 0.96

Tmax 11.82 10.58 11.62 10.50 ---
AUC0-t 33.80 15.70 33.15 15.79 1.02
T1/2 11.23 10.20 11.31 10.26 ---

Explanation of the symbols used –

Cmax - maximal plasma concentration.
C- - minimal plasma concentration.
Cav - average plasma concentration.
tmax - time passed since adminstration at which the
plasma concentration maximum occurs.
AUCt- area under the plasma concentration curve from
administration to time t.
AUC - AUC during a dosage interval to steady state.
MRT - Mean residence time.
T1/2 - plasma concentration half-life.