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l-Thyroxine
[80 KB PDF]
Related Information: Chemical Sampling -
L-thyroxine
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Method no.: |
PV2117 |
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Control no.: |
T-PV2117-01-0303-M |
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Target
concentration: |
30 µg/m³ |
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Procedure: |
Samples are collected by drawing
a known volume of air through a three piece polystyrene cassette containing a
glass fiber filter. Samples are extracted with methyl alcohol and analyzed by
LC using a UV detector. |
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Recommended sampling
time and sampling rate:
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240 min at 1 L/min (240 L)
(maximum flow is 2 L/min for 480 L) |
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Reliable
quantitation limit: |
1.1 µg/m³ |
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Status of method: |
Partially evaluated
method. This method has been subjected to established evaluation procedures of
the Methods Development Team and is presented for information and trial use. |
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Date: |
March 2003 |
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Chemist: |
Mary E. Eide |
|
Methods Development Team
Industrial Hygiene Chemistry Division
OSHA Salt Lake Technical Center
Salt Lake City UT 84070-6406
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1. General Discussion
1.1 Background
1.1.1 History
A request from an OSHA industrial hygienist for a sampling procedure
for l -thyroxine was received at SLTC. l -Thyroxine is a
drug used to treat dysfunctions of the thyroid gland. l -Thyroxine
is prescribed in dosages ranging from 25 to 300 µg,
depending on the person’s weight and the ability of their thyroid
gland tofunction. A target concentration of 30 µg/m³
was based on the maximum recommended dosage of 300 µg
and 10 m³, which is the amount of air the average person
breathes in eight hours. Other hormones have been collected on glass
fiber filters and analyzed by liquid chromatography (LC) using an
ultraviolet detector (see PV2001 Estradiol, etc.)1
A C18 column with a
mobile phase 80:20:0.2 methyl alcohol:DI water:phosphoric acid at
1mL/min gave a good separation of the l -thyroxine peak from the
methyl alcohol. The samples were extracted with methyl alcohol, and had
extraction efficiencies averaging 100.2% for the concentration range of
0.72 to 14.4 µg/filter.
The retention efficiency study showed no l -thyroxine on the back
up filter for two filters in series that had front filters spiked with
14.4 µg, and
had 240-L (at 1 L/min average recovery of 100.4%) or 480-L (at 2 L/min
average recovery of 99.5%) humid air drawn through them. The storage
study showed no loss for samples stored for up to 14 days under both
refrigerated and ambient conditions.
1.1.2 Toxic effects (This section is for information only and should
not be taken as the basis of OSHA policy.)2
l -Thyroxine is used to treat thyroid gland deficiencies.
Overdose symptoms include weight loss, palpitations, nervousness,
diarrhea or abdominal cramps, sweating, tachycardia, cardiac
arrhythmias, angina pectoris, tremors, headache, insomnia, intolerance
to heat, and fever. Prolonged or high overexposure may result in death
due to cardiac arrhythmia or failure.
1.1.3 Workplace exposure3
l -Thyroxine and l -thyroxine sodium are prescribed for over 9
million patients in the United States. Most tablets consumed in the United
States are produced in the United States. Some tablets are exported to other
countries. No current data could be found for the number of workers exposed to l
-thyroxine in the manufacturing process, as the number of companies
manufacturing it is ever-changing due to recent FDA regulations. In August 1997,
FDA announced that all l -thyroxine sodium products were considered new
drugs and must undergo the approval process for new drugs even though they were
currently being sold. At the time this method was written there were only three
forms of l -thyroxine sodium that had completed that process, though old
stocks of unapproved formulations we still being used.
1.1.4 Physical properties and other descriptive information
4,5
CAS number:
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51-48-9
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IMIS:6
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L200
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molecular weight:
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776.93
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melting point:
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223ºC
(decomposes)
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appearance:
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off-white to beige solid
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molecular formula:
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C15H11I4NO4
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solubility:
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water, alcohol, acetone
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|
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synonyms:
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Levothyroxine; l -T4; T4 (hormone);
tetraiodothyronine; 3,3',5,5'-tetraiodo-l
-thyronine; l
-thyroxin; Thyroxinal; Thyroxine; Thyreoideum
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structural formula:
|
|
|
|
I-Thyroxine sodium pentahydrate7 is the alternate form sold in many
pharmaceutical formulations
CAS:
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6106-07-6
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MW:
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888.94
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appearance:
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off-white solid
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|
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solubility:
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water, alcohol, acetone
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|
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synonyms:
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Dathroid;
Eltroxin; Euthyrox; Laevoxin;
Levaxin; Levoroxine;
Levothyroxine sodium; monosodium thyroxine; sodium l -thyroxine;
synthroid; Synthroid R; 3,3',5,5'-tetraiodo-Lthyronine, sodium salt; Thyroxevan;
Unithroid
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|
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structural formula:
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|
This method was evaluated according to the OSHA SLTC "Evaluation
Guidelines for Air Sampling Methods Utilizing Chromatographic Analysis"8.
The Guidelines define analytical parameters, specify required laboratory tests,
statistical calculations and acceptance criteria. The analyte air concentrations
throughout this method are based on the recommended sampling and analytical
parameters.
1.2 Detection limit of the overall procedure (DLOP) and reliable quantitation limit (RQL)
The DLOP is measured as mass per sample and expressed as equivalent air
concentrations, based on the recommended sampling parameters. Ten samplers were
spiked with equal descending increments of analyte, such that the highest
sampler loading was 1.06 µg of l -thyroxine.
This is the amount spiked on a sampler that would produce a peak at least 10
times the response for a sample blank. These spiked samplers were analyzed with
the recommended analytical parameters, and the data obtained used to calculate
the required parameters (standard error of estimate (SEE) and slope) for the
calculation of the DLOP. The slope was 5.18E4 and the SEE was 1169. The RQL is
considered the lower limit for precise quantitative measurements. It is
determined from the regression line parameters obtained for the calculation of
the DLOP, providing 75% to 125% of the analyte is recovered. The DLOP and RQL
were 0.0677 µg and
0.266 µg
respectively. The recovery at the RQL was 99.5%.
Table 1.2
Detection Limit of the Overall Procedure for l
-thyroxine
|
mass per sample
(µg)
|
area counts
(µV·s)
|
|
0.00
0.106
0.212
0.318
0.424
0.530
0.636
0.742
0.848
0.954
1.060
|
0
3806
8629
13406
19021
25092
30573
35053
43231
48901
52931
|
|
|
Figure 1.2. Plot of data to determine the DLOP/RQL for l
- thyroxine at
230 nm. (y=5.18E4x-1927; SEE=1169) |
Below is the chromatogram of the RQL level.
Figure 1.2.2. Chromatogram of the l
- thyroxine peak in a standard near the RQL
at 230 nm. (1=l-thyroxine)
|
2. Sampling Procedure
All safety practices that apply to the work area being sampled should be
followed. The sampling equipment should be attached to the worker in such a
manner that it will not interfere with work performance or safety.
2.1 Apparatus
2.1.1 Samples are collected using a personal sampling pump
calibrated, with the sampling device attached, to within ±5% of the
recommended flow rate.
2.1.2 Samples are collected with three-piece polystyrene cassettes
containing a binderless A/E glass fiber filter. For this evaluation
glass fiber filters were purchased from SKC, Inc. (catalog no. 225-7).
2.2 Reagents
None required.
2.3 Technique
2.3.1 Immediately before sampling, remove the end plugs from the
cassette.
2.3.2 Attach the cassette to the sampling pump so that it is in an
approximately vertical position with the inlet facing up during
sampling. Position the sampling pump, cassette and tubing so it does
not impede work performance or safety.
2.3.3 Air being sampled should not pass through any hose or tubing
before entering the cassette.
2.3.4 After sampling for the appropriate time, remove the cassette,
and replace the top and end plug. Wrap each sample end-to-end with a
Form OSHA-21 seal.
2.3.5 Submit at least one blank sample with each set of samples.
Handle the blank sample in the same manner as the other samples except
draw no air through it.
2.3.6 Record sample volumes (in liters of air) for each sample,
along with any potential interferences.
2.3.7 Ship any bulk samples separate from the air samples.
2.3.8 Submit the samples to the laboratory for analysis as soon as
possible after sampling. If delay is unavoidable, store the samples in
a refrigerator.
2.4 Extraction efficiency
The extraction efficiency of l -thyroxine was determined by
liquid-spiking glass fiber filters with the analyte at 0.1 to 2 times the
target concentration. These samples were stored overnight at ambient
temperature and then extracted and analyzed. The mean extraction
efficiency over the studied range was 100.2%. The wet extraction
efficiency was determined at 1 times the target concentration by liquid
spiking the analyte onto glass fiber filters which had 240-L humid air
(absolute humidity of 15.9 mg/L of water, about 80% relative humidity at
22.2ºC) drawn
through them at 1 L/min immediately before spiking. The mean recovery for
the wet samples was 99.8%.
Table 2.4
Extraction Efficiency (%) of l -Thyroxine
|
level |
sample number |
|
× target
concn |
µg per
sample |
1 |
2 |
3 |
4 |
5 |
6 |
mean |
|
0.1
0.25
0.5
1.0
1.5
2.0
1.0 (wet) |
0.72
1.8
3.6
7.2
10.8
14.4
7.2 |
101.5
99.9
101.4
100.9
101.1
100.4
99.1 |
99.4
100.3
100.3
99.2
100.1
100.2
101.0 |
100.9
101.2
100.4
101.5
100.4
99.3
99.9 |
98.7
100.1
99.5
101.0
99.9
100.6
100.1 |
99.1
99.8
99.9
101.4
100.7
99.9
98.7 |
99.6
98.9
99.3
100.8
100.2
100.6
100.0 |
99.9
100.0
100.1
100.8
100.4
100.2
99.8 |
|
2.5 Retention efficiency
Six glass fiber filters were spiked with 14.4 µg
(60 µg/m³)of l -thyroxine and allowed to equilibrate for 4 h. Each spiked
filter was placed in a cassette and placed in series with a cassette
containing an unspiked glass fiber filter. Each sampling train had 240-L
humid air (absolute humidity of 15.9 mg/L of water, about 80% relative
humidity at 22.2ºC)
pulled through them at 1 L/min. The samples were extracted and analyzed. The
mean recovery was 100.4%. There was no analyte found on the back filters.
Table 2.5.1
Retention Efficiency (%) of l -Thyroxine Sampled
at 1 L/min
|
|
sample number |
|
section |
1 |
2 |
3 |
4 |
5 |
6 |
mean |
|
spiked filter
back up filter
total |
100.8
0.0
100.8 |
101.3
0.0
101.3 |
99.8
0.0
99.8 |
100.2
0.0
100.2 |
99.5
0.0
99.5 |
100.6
0.0
100.6 |
100.4
0.0
100.4 |
|
A test to see if a higher sampling rate could be used was conducted by
spiking six glass fiber filters with 14.4 µg
(60 µg/m³)
of l -thyroxine and allowing them to equilibrate for 4 h. Each spiked
filter was placed in a cassette and placed in series with a cassette containing
an unspiked glass fiber filter. Each sampling train had 480-L humid air
(absolute humidity of 15.9 mg/L of water, about 80% relative humidity at 22.2ºC)
pulled through them at 2 L/min. The samples were extracted and analyzed. The
mean recovery was 99.4%. There was no analyte found on the back filters.
Table 2.5.2
Retention Efficiency (%) of l -Thyroxine Sampled
at 2 L/min
|
|
sample number |
|
section |
1 |
2 |
3 |
4 |
5 |
6 |
mean |
|
spiked filter
back up filter
total |
100.5
0.0
100.5 |
97.6
0.0
97.6 |
100.6
0.0
100.6 |
99.4
0.0
99.4 |
100.2
0.0
100.2 |
98.9
0.0
98.9 |
99.5
0.0
99.5 |
|
2.6 Sample storage
Fifteen glass fiber filters were each spiked with 7.2 µg
(30 µg/m³)
of l -thyroxine. They were allowed to equilibrate for 4 h, then 240-L
of air, with an absolute humidity of 15.7 milligrams of water per liter of
air (about 80% relative humidity at 23ºC),
was drawn through them. Three samples were analyzed immediately. The rest of
the samples were sealed, and six were stored at room temperature (23ºC),
while the other six were stored at refrigerated temperature (4ºC).
Three samples stored at room temperature and three samples stored at
refrigerated temperature were analyzed after 7 days and the remaining
samples after 14 days. The amounts recovered indicate storage was stable at
both temperatures for the time period studied.
Table 2.6
Storage Test for l -thyroxine
|
time
(days) |
ambient storage
recovery (%) |
|
refrigerated storage
recovery (%) |
|
0
7
14 |
101.3
100.8
101.4 |
100.4
100.5
101.1 |
101.5
101.8
100.1 |
|
101.3
101.6
100.4 |
100.4
101.1
101.3 |
101.5
100.9
101.5 |
|
2.7 Recommended air volume and sampling rate
Based on the data collected in this evaluation, 240-L air samples should
be collected at a sampling rate of 1 L/min for 240 minutes, and for greater
sensitivity a higher sampling rate of 2 L/min may be used for 240 minutes
for a 480-L air sample.
2.8 Interferences (sampling)
2.8.1 There are no known compounds which will severely interfere with
the collection of l -thyroxine. Tablets of l -thyroxine
usually contain a filler. If high loadings of l -thyroxine or
l -thyroxine and filler are expected, sample for less time to avoid clogging the filter with the filler.
2.8.2 Suspected interferences should be reported to the laboratory
with submitted samples.
3. Analytical Procedure
Adhere to the rules set down in your Chemical Hygiene Plan. Avoid skin
contact and inhalation of all chemicals and review all appropriate MSDSs.
3.1 Apparatus
3.1.1 A liquid chromatograph equipped with a UV detector. For this
evaluation, a Waters 600 Controller and pump were used, with a Waters
2487 Dual Wavelength Absorbance Detector, and a Waters 717 Plus
Autosampler was used in this evaluation.
3.1.2 An LC column capable of separating l -thyroxine from the
extraction solvent and any potential interferences. A 4.6 × 250 mm
column packed with 5-µm
Bakerbond C18 (JT Baker, Phillipsburg, NJ) was used in this evaluation.
3.1.3 An electronic integrator or some other suitable means of
measuring peak areas. A Waters Millennium32 Data System was
used in this evaluation.
3.1.4 Glass vials with poly(tetrafluoroethylene)-lined caps. For this
evaluation 4-mL vials were used.
3.1.5 A dispenser capable of delivering 2.0 mL of extracting solvent
to prepare standards and samples. If a dispenser is not available, a
2.0-mL volumetric pipet may be used.
3.1.6 Volumetric flasks - 10-mL and other convenient sizes for
preparing standards.
3.2 Reagents
3.2.1 l -Thyroxine sodium. Spectrum 99-103% (lot QK0730) was
used in this evaluation.
3.2.2 Methyl alcohol, HPLC grade. Fisher 99.9% (lot 023066) was used
for this evaluation.
3.2.3 Deionized water (DI water). A Barnstead NANOpure Diamond water
deionizer was used in this evaluation.
3.2.4 Mobile phase was 80:20:0.2 methyl alcohol: DI water:phosphoric
acid.
3.3 Standard preparation
3.3.1 Freshly prepare two stock standards. A stock standard may be
prepared by weighing out 28.8 mg of l -thyroxine in a 10-mL
flask, then fill to the mark with methyl alcohol.
3.3.2 Diluted standards are prepared by serial dilution with methyl
alcohol. Bracket sample concentrations with working standard
concentrations. If sample concentrations are higher than the
concentration range of prepared standards, either analyze higher
standards, or dilute the sample. The higher standards should be at least
as high in concentration as the highest sample. The range of standards
used in this study was from 0.1 to 28.8 µg/mL.
3.4 Sample preparation
3.4.1 Open the cassette and carefully transfer the glass fiber filter
to a labeled 4-mL vial. Wipe the inside of the cassette with a glass
fiber filter and place into a separate labeled 4-mL vial.
3.4.2 Add 2.0 mL of methyl alcohol to each vial using the same
dispenser as used for preparation of standards or use a volumetric pipet.
3.4.3 Immediately seal the vials with poly(tetrafluoroethylene)-lined
caps.
3.4.4 Shake the vials on a shaker, or rotate on a rotator, for 30 minutes.
3.5 Analysis
3.5.1 HPLC conditions
column:
|
Bakerbond C18 column 4.6 × 250 mm
|
Figure 3.5.1. A chromatogram of 14.4 ug/mL l - thyroxine in methyl
alcohol at 230 nm. Key:(1) methyl alcohol; (2) l -
thyroxine.
|
injection size:
|
10 µL
|
mobile phase:
|
1 mL/min 80:20:0.2 methyl alcohol:
water: phosphoric acid
|
detector:
|
UV at 230 and 254 nm
|
run time:
|
15 min
|
retention times:
|
2.6 min methyl alcohol
9.8 min l -thyroxine
|
|
|
|
|
3.5.2 Peak areas are measured by an integrator or other suitable
means.
3.5.3 An external standard (ESTD) calibration method is used. A
calibration curve can be constructed by plotting response of standard
injections versus micrograms of analyte per sample. Bracket the samples
with freshly prepared analytical standards over a range of
concentrations.
|
Figure 3.5.2. Calibration curve of l - thyroxine at 230 nm.
(Y=2.74E4x - 1.2.3E4) |
3.6 Interferences (analytical)
3.6.1 Any compound that produces an LC response
and has a similar retention time as the analyte is a potential
interference. If any potential interferences were reported, they should
be considered before samples are extracted. Generally, chromatographic
conditions can be altered to separate an interference from the analyte.
3.6.2 When necessary, the identity or purity of an analyte peak may
be confirmed by a photodiode array scan of the peak, by wavelength
ratioing, or by LC/mass spec. |
Figure 3.6.2. The UV spectrum of l - thyroxine in methyl alcohol |
3.7 Calculations
The amount of analyte per sampler, including the amount found in the
cassette wipe, is obtained from the appropriate calibration curve in terms
of micrograms per sample, uncorrected for extraction efficiency. This total
amount is then corrected by subtracting the total amount (if any) found on
the blank. The air concentration is calculated using the following formulas.
|
where |
CM is concentration by weight (mg/m³)
M is micrograms per sample
V is liters of air sampled
EE is extraction efficiency, in decimal form |
4. Recommendations for further study
Collection, reproducibility, and other detection limit studies need to be
performed to make this a validated method.
1Sampling and Analytical Methods.
(accessed 6/24/2002).
2Arky, R., Physicians’ Desk Reference, Medical Economics Company:
Montvale, NJ, 1997, p 1015.
3
WebMD.
(accessed 6/25/02).
4Lewis, R., Sax’s Dangerous Properties of Industrial Materials, Van
Nostrand Reinhold: New York, 2002, p 176.
5
CHEMINFO Database
(accessed 5/21/2002).
6
Chemical Sampling Information (CSI)
(accessed 5/21/2002).
7MSDS Sigma-Aldrich http://www.sigmaaldrich.com (accessed 5/21/2002).
8Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. C. Evaluation Guidelines for Air Sampling Methods Utilizing Chromatographic Analysis;
OSHA
Salt Lake Technical Center, U.S. Department of Labor: Salt Lake City, UT, 1999.
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