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__________________________________

Compartment [FNa] Form Ingredient Quantity [FNb]
_______________________________________________________________________________
#2,3, & 4 Liquid Picrate 0.11 mmol
#6 Liquid NaOH (for pH
adjustment) [FNc]
_______________________________________________________________________________
[FNa]. Compartments are numbered 1-7, with compartment #7 located closest to
pack fill position #2.
[FNb]. Nominal value at manufacture.
[FNc]. See PRECAUTIONS.




PRECAUTIONS: COMPARTMENT #6 CONTAINS 75 m L OF 10 N NaOH; AVOID CONTACT; SKIN IRRITANT; RINSE CONTACTED AREA WITH WATER.
USED PACKS CONTAIN HUMAN BODY FLUIDS; HANDLE WITH APPROPRIATE CARE.
FOR IN VITRO DIAGNOSTIC USE.
MIXING & DILUTING: Mixing and diluting are automatically performed by the ACA [ discrete clinical analyzer. The sample cup must contain sufficient quantity to accommodate the sample volume plus the "dead volume"; precise cup filling is not required.
Sample Cup Volumes ( m L)


________________________________________
Standard Microsystem
Analyzer Dead Total Dead Total
________________________________________
II, III 120 3000 10 500
IV, SX 120 3000 30 500
V 90 3000 10 500


STORAGE OF UNPROCESSED PACKS: Store at 2-8 ° C. Do not freeze. Do not expose to temperatures above 35 ° C or to direct sunlight.
EXPIRATION: Refer to EXPIRATION DATE on the tray label.
SPECIMEN COLLECTION: Serum or urine can be collected and stored by normal procedures. [FN2]
KNOWN INTERFERING SUBSTANCES [FN3]: Serum Protein Influence. Serum protein levels exert a direct influence on the CREA assay. The following should be taken into account when this method is used for urine samples and when it is calibrated:
Aqueous creatinine standards or urine specimens will give CREA results depressed by approximately 0.7 mg/dL [62 m mol/L] [FNd] and will be less precise than samples containing more than 3 g/dL [30 g/L] protein.
All urine specimens should be diluted with an albumin solution to give a final protein concentration of at least 3 g/dL [30 g/L]. Du Pont Enzyme Diluent (Cat. #790035-901) may be used for this purpose.
<> High concentration of endrogenous bilirubin (>20 mg/dL [>342 m mol/L]) will give depressed CREA results (average depression 0.8 mg/dL [71 m mol/L]). [FN4]
<> Grossly hemolyzed (hemoglobin100 mg/dL [>62 mol/L]) or visibly lipemic specimens may cause falsely elevated CREA results. [FN5], [FN6]
<> The following cephalosporin antibiotics do not interfere with the CREA method when present at the concentrations indicated. Systematic inaccuracies (bias) due to these substances are less than or equal to 0.1 mg/dL [8.84 m mol/L] at CREA concentrations of approximately 1 mg/dL [88 m mol/L].

Peak Drug
Serum Level [FN7] , [FN8], [FN9] Concentration
Antibiotic mg/oL [mmol/L] mg/dL [mmol/L]
Cephaloridine 1.4 0.3 25 6.0
Cephalexin 0.6-2.0 0.2-0.6 25 7.2
Cephamandole 1.3-2.5 0.3-0.5 25 4.9
Cephapirin 2.0 0.4 25 5.6
Cephradine 1.5-2.0 0.4-0.6 25 7.1
Cefazolin 2.5-5.0 0.55-1.1 50 11.0


<> The following cephalosporin antibiotics have been shown to affect CREA results when present at the indicated concentrations. System inaccuracies (bias) due to these substances are greater that 0.1 mg/dL [8.84 mol/L] at CREA concentrations of: The following cephalosporin antibiotics have been shown to affect CREA results when present at the indicated concentrations. System inaccuracies (bias) due to these substances are greater that 0.1 mg/dL [8.84 mol/L] at CREA concentrations of:


Peak Drug
Serum Level [FN7], [FN8], Concentration Effect
[FN9]>
Antibiotic mg/dL [mmol/L] mg/dL [mmol/L]
Cephalothin 1-6 0.2-1.5 100 25.2 <>
20-25%
Cephoxitin 2.0 0.5 5.0 1.2 <>
35-40%


<> The single wavelength measurement used in this method eliminates interference from chromophores whose 510 nm absorbance is constant throughout the measurement period.
[FNd]. Systeme International d'unites (S.I. Units) are in brackets.
<> Each laboratory should determine the acceptability of its own blood collection tubes and serum separation products. Variations in these products may exist between manufacturers and, at times, from lot to lot.

PROCEDURE:
TEST MATERIALS


________________________________________________
II,III IV,SX V
Du Pont Du Pont Du Pont
Item Cat.# Cat.# Cat. #
________________________________________________
ACA [ CREA
Analytical
Test Pack 701976901 701976901 701976901
Sample System
Kit 710642901 710642901 713697901
or
Micro Sample
System Kit 702694901 710356901 NA
and
Micro Sample

System
Holders 702785000 NA NA
DYLUX [
Photosensitive
Printer
Paper 700036000 NA NA
Thermal Printer
Paper NA 710639901 713645901
Du Pont
Purified
Water 704209901 710615901 710815901
Cell Wash
Solution 701864901 710664901 710864901
________________________________________________


TEST STEPS
The operator need only load the sample kit and appropriate test pack(s) into a properly prepared ACA [ discrete clinical analyzer. It automatically advances the pack(s) through the test steps and prints a result(s). See the Instrument Manual of the ACA [ analyzer for details of mechanical travel of the test pack(s).
<> Preset Creatinine (CREA) Test Conditions
<> Sample Volume: 200 m L
<> Diluent: Purified Water
<> Temperature: 37.0 + 0.1 <> C
<> Reaction Period: 29 seconds
<> Type of Measurement: Rate
<> Measurement Period: 17.07 seconds
<> Wavelength: 510 nm
<> Units: mg/dL [ m mol/L]

CALIBRATION
The general calibration procedure is described in the Calibration/Verification chapter of the Manuals. The following information should be considered when calibrating the CREA method.
<> Assay Range: 0-20 mg/mL[0-1768 m mol/L] [FNe]
<> Reference Material: Protein containing primary standards [FNf] or secondary calibrators such as Du Pont Elevated Chemistry Control (Cat. #790035903) and Normal Chemistry Control (Cat. #790035905) [FNg]
<> Suggested Calibration levels: 1,5,20, mg/mL [88, 442, 1768 m mol/L]
<> Calibration Scheme: 3 levels, 3 packs per level
<> Frequency: Each new pack lot. Every 3 months for any one pack lot.
[FNe]. For the results in S.I. units [ m mol/L] the conversion factory is 88.4.

[FNf]. Refer to the Creatinine Standard Preparation and Calibration Procedure available on request from a Du Pont Representative.
[FNg]. If the Du Pont Chemistry Controls are being used, prepare them according to the instructions on the product insert sheets.
Preset Creatinine (CREA) Test Conditions


_______________________________________________________________________________
_______________________________________________________________________________
ACA[ ACA [
II III, IV, SX, V
Item Analyzer Analyzer
_______________________________________________________________________________
_______________________________________________________________________________
Count by One(1) NA
[Five (5)]
Decimal
Point 0.0 mg/dL 000.0 mg/dL
Location [000.<>mol/L] [000<>mol/L]

Assigned
Starting 999.8 -1.000 E1
Point or
Offset C 0 [9823.] [-8.840 E2]
Scale Factor
or 0.2000 2.004 E-1 [FNh]
Assigned mg/dL/count [FNh]
Linear Term
C 1 [FNh] [0.3536 [1.772E1]
m mol/L/count]
_______________________________________________________________________________
_______________________________________________________________________________
[FNh.] The preset scale factor(linear term) was derived from the molar
absorptivity of the indicator and is based on an absorbance to activity
relationship (sensitivity) of 0.596 (mA/min)/(<>/L). Due to small
differences in filters and electronic components between instruments, the
actual scale factor (linear term) may differ slightly from that given above.


QUALITY CONTROL

Two types of quality control procedures are recommended:
<> General Instrument Check. Refer to the Filter Balance Procedure and the Absorbance Test Method described in the Instrument Manual. Refer also to the ABS Test Methodology literature.
<> Creatinine Method Check. At least once daily run a CREA test on a solution of known creatinine activity such as an assayed control or calibration standard other than that used to calibrate the CREA method. For further details review the Quality Assurance Section of the Chemistry Manual. The result obtained should fall within acceptable limits defined by the day-to-day variability of the system as measured in the user's laboratory. (See SPECIFIC PERFORMANCE CHARACTERISTICS for guidance.) If the result falls outside the laboratory's acceptable limits, follow the procedure outlined in the Chemistry Troubleshooting Section of the Chemistry Manual.
A possible system malfunction is indicated when analysis of a sample with five consecutive test packs gives the following results:

Level SD

1 mg/dL >0.15 mg/dL
[88<>mol/L] [>13 m mol/L]
20 mg/dL >0.68 mg/dL
[1768 m mol/L] [>60 m mol/L]


Refer to the procedure outlined in the Trouble Shooting Section of the Manual.
RESULTS
The ACA [ analyzer automatically calculates and prints the CREA result in mg/dL [ m mol/L].
LIMITATION OF PROCEDURE
Results >20 mg/dL [1768 m mol/L]:
<> Dilute with suitable protein base diluent. Reassay. Correct for diluting before reporting.
The reporting system contains error messages to warn the operator of specific malfunctions. Any report slip containing a letter code or word immediately following the numerical value should not be reported. Refer to the Manual for the definition of error codes.
REFERENCE INTERVAL
SERUM: [FN11], [FNi] Males - 0.8-1.3 md/dL[71-115 mmol/L] ] Females - 0.6-1.0 md/dL [53-88 mmol/L]
URINE [FN12]
Males - 0.6-2.5 g/24 hr [53-221 mmol/24 hr]Females - 0.6-1.5 g/24 hr [53-133 mmol/24 hr]
Each laboratory should establish its own reference intervals for CREA as performed on the analyzer.
[FNi]. Reference interval data obtained from 200 apparently healthy individuals (71 males, 129 females) between the ages of 19 and 72.
SPECIFIC PERFORMANCE CHARACTERISTICS
REPRODUCIBILITY [FNK]


Standard Deviation (% CV)
Material Mean Within-Run Between-Day
________________________________________________
Lyophilized 1.3 0.05 (3.7) 0.05 (3.7)
Control [115] [4.4] [4.4]
Lyophilized 20.6 0.12 (0.6) 0.37 (1.8)
Control [1821] [10.6] [32.7]
________________________________________________


CORRELATION Regression Statistics [FNl]


_______________________________________________________________________________
Comparative Correlation
Method Slope Intercept Coefficient n
_______________________________________________________________________________

Autoanalyzer [ 1.03 0.03[2.7] 0.997 260
_______________________________________________________________________________
[FNj.] ALL SPECIFIC PERFORMANCE CHARACTERISTICS tests were run after normal
recommended equipment quality control checks were performed (see Instrument
Manual).
[FNk.] Specimens at each level were analyzed in duplicate for twenty days. The
within-run and between-day standard deviations were calculated by the
analysis of variance method.
[FNl.] Model equation for regression statistics is: Result of ACA [ Analyzer =
Slope Comparative method result) + intercept


ASSAY RANGE [FNm]
0.0-20.0 mg/dl [0-1768 m mol]
[FNm]. See REPRODUCIBILITY for method performance within the assay range.
ANALYTICAL SPECIFICITY
See KNOWN INTERFERING SUBSTANCES section for details.

BIBLIOGRAPHY:
[FN1] Larsen, K, Clin Chem Acta 41, 209 (1972).
[FN2] Tietz, NW, Fundamentals of Clinical Chemistry, W. B. Saunders Co., Philadelphia, PA, 1976, pp 47-52, 1211.
[FN3] Supplementary information pertaining to the effects of various drugs and patient conditions on in vivo or in vitro diagnostic levels can be found in "Drug Interferences with Clinical Laboratory Tests," Clin. Chem 21 (5) (1975), and "Effects of Disease on Clinical Laboratory Tests," Clin Chem, 26 (4) 1D-476D (1980).
[FN4] Watkins, R. Fieldkamp, SC, Thibert, RJ, and Zak, B, Clin Chem, 21, 1002 (1975).
[FN5] Kawas, EE, Richards, AH, and Bigger, R, An Evaluation of a Kinetic Creatinine Test for the Du Pont ACA, Du Pont Company, Willmington, DE (February 1973). [FNn]

[FN6] Westgard, JO, Effects of Hemolysis and Lipemia on ACA Creatinine Method,0.200 m L, Sample Size, Du Pont Company, Wilmington, DE (October 1972).
[FN7] Physicians' Desk Reference, Medical Economics Company, 33 Edition, 1979.
[FN8] Henry, JB, Clinical Diagnosis and Management by Laboratory Methods, W.B. Saunders Co., Philadelphia, PA 1979, Vol. III.
[FN9] Krupp, MA, Tierney, LM Jr., Jawetz, E, Roe, Rl, Camargo, CA, Physicians Handbook, Lange Medical Publications, Los Altos, CA, 1982 pp 635- 636.
[FN1] 0 Sarah, AJ, Koch, TR, Drusano, GL, Celoxitin Falsely Elevates Creatinine Levels, JAMA 247, 205-206 (1982).
[FN1] 1 Gadsden, RH, and Phelps, CA, A Normal Range Study of Amylase in Urine and Serum on the Du Pont ACA, Du Pont Company, Wilmington, DE (March 1978) [FNn]
[FN1] 2 Dicht, JJ, Reference Intervals for Serum Amylase and Urinary Creatinine on the Du Pont ACA [ Discrete Clinical Analyzer, Du Pont Company, Wilimgton, DE (November 1984).
[FNn]. Reprints available from Du Pont Company, Diagonostic Systems.
Attachment 3
Analysis of Creatinine for the Normalization of Cadmium and Beta-2- Microglobulin Concentrations in Urine
Matrix: Urine
Target Concentration: 1.1 g/L (this amount is representative of creatinine concentrations found in urine).
Procedure: A 1.0 mL aliquot of urine is passed through a C18 SEP-PAK (Waters Associates). Approximately 30 mL of HPLC (high performance liquid chromatography) grade water is then run through the SEP-PAK. The resulting solution is diluted to volume in a 100-mL volumetric flask and analyzed by HPLC using an ultraviolet (UV) detector.

Special Requirements: After collection, samples should be appropriately stabilized for cadmium (Cd) analysis by using 10% high purity (with low Cd background levels) nitric acid (exactly 1.0 mL of 10% nitric acid per 10 mL of urine) or stabilized for Beta-2-Microglobulin (B2M) by taking to pH 7 with dilute NaOH (exactly 1.0 mL of 0.11 N NaOH per 10 mL of urine). If not immediately analyzed, the samples should be frozen and shipped by overnight mail in an insulated container.
Date: January 1992.
Chemists: David B. Armitage, Duane Lee, Organic Service Branch II, OSHA Technical Center, Salt Lake City, Utah.
1. General Discussion
1.1. Background
1.1.1. History or procedure
Creatinine has been analyzed by several methods in the past. The earliest methods were of the wet chemical type. As an example, creatinine reacts with sodium picrate in basic solution to form a red complex, which is then analyzed colorimetrically (Refs. 5.1 and 5.2).
Since industrial hygiene laboratories will be analyzing for Cd and B2M in urine, they will be normalizing those concentrations to the concentration of creatinine in urine. A literature search reveled several HPLC methods (Refs. 5.3., 5.4., 5.5. and 5.6) for creatinine in urine and because manu industrial hygiene laboratories have HPLC equipment, it was desirable to develop an industrial hygiene HPLC method for creatinine in urine. The method of Hausen, Fuchs, and Wachter was chosen as the starting point for method development, SEP-PAKs were used for sample clarification and cleanup in this method to protect the analytical column. The urine aliquot which has been passed through the SEP-PAK is then analyzed by reverse-phase HPLC using ion-pair techniques.
This method is very similar to that of Ogata and Taguchi (Ref. 5.6.) except they used centrifugation for sample clean-up. It is also of note that they did a comparison of their HPLC results to those of the Jaffe method (a picric acid method commonly used in the health care industry) and found a linear relationship of close to 1:1. This indicates that either HPLC or colorimetric methods may be used to measure creatinine concentrations in urine.
1.1.2. Physical properties (Ref 5.7.)
Molecular weight: 113.12
Molecular formula: C 4 -H 7 -N 3 -O
Chemical name: 2-amino-1.5-dihydro-1-methyl-4H-imidazol-4- one
CAS#: 60-27-5
Melting point: 300>C (decomposes)
Appearance: white powder
Solubility: soluble in water; slightly soluble in alcohol; practically insoluble in acetone, ether, and chloroform
Synonyms: 1-methylglycocyamidine, 1-methylhydation-2- imide
Structure: see Figure #1
Figure #1
Figure #1
1.2. Advantages
1.2.1. This method offers a simple, straightforward, and specific alternative method to the Jaffe method.
1.2.2. HPLC instrumentation is commonly found in many industrial hygiene laboratories.
2. Sample Stabilization Procedure
2.1. Apparatus Metal-free plastic container for urine sample.
2.2. Reagents
2.2.1. Stabilizing Solution-1) Nitric acid (10%, high purity with low Cd background levels) for stabilizing urine for Cd analysis or 2) NaOH, 0.11 N, for stabilizing urine for B2M analysis.
2.2.2. HPLC grade water
2.3. Technique
2.3.1. Stabilizing solution is added to the urine sample (see section 2.2.1.) The stabilizing solution should be such that for each 10 mL of urine, add exactly 1.0 mL of stabilizer solution. (Never add water or urine to acid or base. Always add acid or base to water or urine.) Exactly 1.0 mL of 0.11 N NaOH added to 10 mL of urine should result in a pH of 7. Or add 1.0 mL of 10% nitric acid to 10 mL of urine.
2.3.2. After sample collection seal the plastic bottle securely and wrap it with an appropriate seal. Urine samples should be frozen and then shipped by overnight mail (if shipping is necessary) in an insulted container. (Do not fill plastic bottle too full. This will allow for expansion of contents during the freezing process.)
2.4. The Effect of Preparation and Stabilization Techniques on Creatinine Concentrations
Three urine samples were prepared by making one sample acidic, not treating a second sample, and adjusting a third sample to pH 7. The samples were analyzed in duplicate by two different procedures. For the first procedure a 1.0 mL aliquot of urine was put in a 100-mL volumetric flask, diluted to volume with HPLC grade water, and then analyzed directly on an HPLC. The other procedure used SEP-PAKs. The SEP-PAK was rinsed with approximately 5 mL of methanol followed by approximately 10 mL of HPLC grade water and both rinses were discarded. Then 1.0 mL of the urine sample was put through the SEP-PAK, followed by 30 mL of HPLC grade water. The urine and water were transferred to a 100-mL volumetric flask, diluted to volume with HPLC grade water and analyzed by HPLC. These three urine samples were analyzed on the day they were obtained and then frozen. The results show that whether the urine is acidic, untreated or adjusted to pH 7, the resulting answer for creatinine is essentially unchanged. The purpose of stabilizing the urine by making it acidic or neutral is for the analysis of Cd or B2M respectively.
COMPARISON OF PREPARATION AND STABILIZATION TECHNIQUES


Sample w/o SEP-PAK with SEP-PAK
(g/L creatinine) (g/L creatinine)
_____________________________________________
Acid 1.10 1.10
Acid 1.11 1.10
Untreated 1.12 1.11
Untreated 1.11 1.12

pH 7 1.08 1.02
pH 7 1.11 1.08
_____________________________________________


2.5. Storage After 4 days and 54 days of storage in a freezer, the samples were thawed, brought to room temperature and analyzed using the same procedures as in section 2.4. The results of several days of storage show that the resulting answer for creatinine is essentially unchanged.
Storage Data


-------------------------------------------------------------------------------
4 days 54 days
------------------------------------------------------------
w/o SEP- with SEP- w/o SEP- with SEP-
Sample PAK (g/L PAK (g/L PAK (g/L PAK (g/L
creatinine) creatinine)
creatinine) creatinine)
-------------------------------------------------------------------------------

Acid............. 1.09 1.09 1.08 1.09
Acid............. 1.10 1.10 1.09 1.10
Acid............. ............................ ......... 1.09 1.09
Untreated........ 1.13 1.14 1.09 1.11
Untreated....1.15 1.14 1.10 1.10
Untreated........ ............................ ......... 1.09 1.10
pH 7............. 1.14 1.13 1.12 1.12
pH 7............. 1.14 1.13 1.12 1.12
pH 7............. ............................ ......... 1.12 1.12
-------------------------------------------------------------------------------




2.6. Interferences: None.
2.7. Safety precautions
2.7.1. Make sure samples are properly sealed and frozen before shipment to avoid leakage.
2.7.2. Follow the appropriate shipping procedures.
The following modified special safety precautions are based on those recommended by the Centers for Disease Control (CDC) (Ref.5.8).
2.7.3. Wear gloves, lab coat, and safety glasses while handling all human urine products. Disposable plastic, glass, and paper (pipet tips, gloves, etc.) that contact urine should be placed in a biohazard autoclave bag. These bags should be kept in appropriate containers until sealed and autoclave. Wipe down all work surfaces with 10% sodium hypochlorite solution when work is finished.
2.7.4. Dispose of all biological samples and diluted specimens in a biohazard autoclave bag at the end of the analytical run.
2.7.5. Special care should be taken when handling and dispensing nitric acid. Always remember to add acid to water (or urine). Nitric acid is a corrosive chemical capable of severe eye and skin damage. Wear metal-free gloves, a lab coat, and safety glass. If the nitric acid comes in contact with any part of the body, quickly wash with copious quanties of water for at least 15 minutes.
2.7.6. Special care should be taken when handling and dispensing NaOH. Always remember to add base to water (or urine). NaOH can cause severe eye and skin damage. Always wear the appropriate gloves, a lab coat, and safety glasses. If the NaOH comes in contact with any part of the body, quickly wash with copius quantities of water for at least 15 minutes.
3. Analytical Procedure
3.1. Apparatus
3.1.1. A high performance liquid chromatograph equipped with pump, sample injector and UV dector.
3.1.2. A C18 HPLC column; 25 cm X 4.6 mm I.D.
3.1.3. An electronic integrator, or some other suitable means of determining analyte response.
3.1.4. Stripchart recorder.
3.1.5. C18 SEP-PAKs (Waters Associates) or equivalent.
3.1.6. Luer-lock syringe for sample preparation (5 mL or 10 mL).
3.1.7. Volumetric pipettes and flasks for standard and sample preparation.
3.1.8. Vacuum system to aid sample preparation (optional).
3.2. Reagents
3.2.1. Water, HPLC grade.
3.2.2. Methanol, HPLC grade.
3.2.3. PIC B-7 (Waters Associates) in small vials.
3.2.4. Creatinine, anhydrous, Sigma Chemical Corp., purity not listed.
3.2.5. 1-Heptanesulfonic acid, sodium salt monohydrate.
3.2.6. Phosphoric acid.
3.2.7. Mobile phase. It can be prepared by mixing one vial of PIC B-7 into a 1 L solution of 50% methanol and 50% water. The mobil phase can also be made by preparing a solution that is 50% methanol and 50% water with 0.005M heptanesulfonic acid and adjusting the pH of the solution to 3.5 with phosphoric acid.
3.3. Standard preparation
3.3.1. Stock standards were prepared by weighing 10 to 15 mg of creatinine. This is transferred to a 25-mL volumetric flask and diluted to volume with HPLC grade water.
3.3.2. Dilutions to a working range of 3 to 35 g/mL are made in either HPLC grade water or HPLC mobil phase (standards give the same detector response in either solution).
3.4. Sample preparation
3.4.1. The C18 SEP-PAK is connected to a Luer-lock syringe. It is rinsed with 5 mL HPLC grade methanol and then 10 mL of HPLC grade water. These rinses are discarded.
3.4.2. Exactly 1.0 mL of urine is pipetted into the syringe. The urine is put through the SEP-PAK into a suitable container using a vacuum system.
3.4.3. The walls of the syringe are rinsed in several stages with a total of approximately 30 mL of HPLC grade water. These rinses are put through the SEP-PAK into the same container. The resulting solution is transferred to a 100-mL volumetric flask and then brought to volume with HPLC grade water.
3.5. Analysis (conditions and hardware are those used in this evaluation.)
3.5.1. Instrument conditions
Column Zorbax V ODS, 5-6 m m particle size; 25 cm X 4.6 mm I.D.
Mobile phase See Section 3.2.7.
Detector Dual wavelength UV; 229 nm (primary) 254 nm (secondary),
Flow rate 0.7 mL/minute.
Retention time 7.2 minutes.
Sensitivity 0.05 AUFS.
Injection volume 20 m L.
3.5.2. Chromatogram (See Figure #2).


3.6. Interferences
3.6.1. Any compound that has the same retention time as creatinine and absorbs at 229 nm is an interference.
3.6.2. HPLC conditions may be varied to circumvent interferences. In addition, analysis at another UV wavelength (i.e. 254 nm) would allow a comparison of the ratio of response of a standard to that of a sample. Any deviations would indicate an interference.
3.7. Calculations
3.7.1. A calibration curve is constructed by plotting dector response versus standard concentration (See Figure #3).
3.7.2. The concentration of creatinine in a sample is determined by finding the concentration corresponding to its detector response. (See Figure #3).


3.7.3. The m g/mL creatinine from section 3.7.2. is then multiplied by 100 (the dilution factor). This value is equivalent to the micrograms of creatinine in the 1.0 mL stabilized urine aliquot or the milligrams of creatinine per liter of urine. The desire unit, g/L, is determined by the following relationship:
3.7.4. The resulting value for creatinine is used to normalize the urinary concentration of the desired analyte (A) (Cd or B2M) by using the following formula.
Where A is the desired analyte. The protocol of reporting such normalized results is m g A/g creatinine.
3.8. Safety precautions. See section 2.7
4. Conclusions
The determination of creatinine in urine by HPLC is a good alternative to the Jaffe method for industrial hygiene laboratories. Sample clarification with SEP-PAKs did not change the amount of creatinine found in urine samples. However, it does protect the analytical column. The results of this creatinine in urine procedure are unaffected by the pH of the urine sample under the conditions tested by this procedure. Therefore, no special measure are required for creatinine analysis whether the urine sample has been stabilized with 10% nitric acid for the Cd analysis or brought to a pH of 7 with 0.11 NaOH for the B2M analysis.
5. References
5.1. Clark, L.C.; Thompson, H.L. Anal. Chem. 1949, 21, 1218.
5.2. Peters, J.H; J. Biol. Chem. 1942, 146, 176.
5.3. Hausen. V.A.; Fuchs, D; Wachter, H.;J. Clin. Chem. Clin. Biochem. 1981, 19,373-378.
5.4. Clark, P.M.S.; Kricka, L.J.; Patel, A.; J. Liq. Chrom. 1980, 3(7), 1031- 1046.
5.5. Ballerini, R.; Chinol, M.; Cambi, A; J. Chrom. 1979, 179, 365-369.
5.6. Ogata, M.; Taguchi, T.; Industrial Health 1987, 25, 225-228.
5.7. "Merck Index", 11th ed.; Windholz, Martha Ed.; Merck: Rahway, N.J., 1989; p. 403.
5.8. Kimberly, M.; "Determination of Cadmium in Urineby Graphite Furance Atomic Absorption Spectrometry with Zeeman Background Correction." Centers for Disease Control, Atlanta, Georgia, unpublished, update 1990.


Note: Authority cited: Sections 142.3, 9020, 9030 and 9040, Labor Code. Reference: Sections 142.3, 9004(d), 9009, 9020, 9030, 9031 and 9040, Labor Code.






s 5208. Asbestos.
(a) Scope and application -
(1) This section applies to all occupational exposures to asbestos in all industries covered by the California Occupational Safety and Health Act, except as provided in subsection (a)(2) and (3) of this section.

(2) This section does not apply to construction work as defined in Section 1502, except for the spraying of asbestos containing material in subsection 5208(f)(1)(J). (Exposure to asbestos in construction work other than spraying is covered by Section 1529).
(3) This section does not apply to ship repairing, shipbuilding and shipbreaking employments and related employments as defined in Section 8354: Definitions. (Exposure to asbestos in these employments is covered by Section 8358).
(4) Whenever employee exposure to asbestos as defined in subsection (b) of this section consists only of exposure to tremolite, anthophyllite, and actinolite in the non-asbestiform mineral habit, the provisions of section 5208.1 shall apply and supersede the provisions of this section.
(5) The provisions of this section are subject to the requirements of the Occupational Carcinogen Control Act of 1976 (Labor Code, Division 5, Part 10).
(b) Definitions -
"Asbestos" includes chrysotile, amosite, crocidolite, tremolite asbestos, anthophyllite asbestos, actinolite asbestos, and any of these minerals that have been chemically treated and/or altered.
"Asbestos-containing material (ACM)" means any material containing more than 1% asbestos.
"Chief" means the Chief of the Division of Occupational Safety and Health, California Department of Industrial Relations.
"Authorized person" means any person authorized by the employer and required by work duties to be present in regulated areas.
"Building/facility owner" is the legal entity, including a lessee, which exercises control over management and record keeping functions relating to a building and/or facility in which activities covered by this standard take place.
"Certified Industrial Hygienist (CIH)" means one certified in the practice of industrial hygiene by the American Board of Industrial Hygiene.
"Director" means the Director of the National Institute for Occupational Safety and Health, U.S. Department of Health and Human Services, or designee.
"Employee exposure" means that exposure to airborne asbestos that would occur if the employee were not using respiratory protective equipment.
"Fiber" means a particulate form of asbestos 5 micrometers or longer, with a length-to-diameter ratio of at least 3 to 1.
"High-efficiency particulate air (HEPA) filter" means a filter capable of trapping and retaining at least 99.97 percent of 0.3 micrometer diameter monodisperse particles.
"Homogeneous area" means an area of surfacing material or thermal system insulation that is uniform in color and texture.
"Incident" means any unanticipated event which causes, or is immediately likely to cause, an exposure of an employee, unprotected by an appropriate respirator, to asbestos fibers in excess of the permissible exposure limit.
"Industrial hygienist" means a professional qualified by education, training, and experience to anticipate, recognize, evaluate and develop controls for occupational health hazards.
"PACM" means "presumed asbestos containing material".
"Presumed Asbestos Containing Material" means thermal system insulation and surfacing material found in buildings constructed no later than 1980. The designation of a material as "PACM" may be rebutted pursuant to subsection (j)(8) of this section.
"Regulated area" means an area established by the employer to demarcate areas where airborne concentrations of asbestos exceed, or there is a reasonable possibility they may exceed, the permissible exposure limits.
"Surfacing ACM" means surfacing material which contains more than 1% asbestos.
"Surfacing materials" means material that is sprayed, troweled-on or otherwise applied to surfaces (such as acoustical plaster on ceilings and fireproofing materials on structural members, or other materials on surfaces for acoustical, fireproofing, and other purposes).
"Thermal System Insulation (TSI)" means ACM applied to pipes, fittings, boilers, breeching, tanks, ducts or other structural components to prevent heat loss or gain.
"Thermal System Insulation ACM" means thermal system insulation which contains more than 1% asbestos.
(c) Permissible exposure limit (PELS) -
(1) Time-weighted average limit (TWA): The employer shall ensure that no employee is exposed to an airborne concentration of asbestos in excess of 0.1 fiber per cubic centimeter (0.1 f/cc) of air as an eight (8)-hour time-weighted average (TWA) as determined by the method prescribed in Appendix A to this section, or by an equivalent method.
(2) Excursion limit: The employer shall ensure that no employee is exposed to an airborne concentration of asbestos in excess of 1.0 fiber per cubic centimeter of air (1 f/cc) as averaged over a sampling period of thirty (30) minutes as determined by the method prescribed in Appendix A to this section, or by an equivalent method.
(d) Exposure monitoring -
(1) General.
(A) Determinations of employee exposure shall be made from breathing zone air samples that are representative of the 8-hour TWA and 30-minute short-term exposures of each employee.
(B) Representative 8-hour TWA employee exposures shall be determined on the basis of one or more samples representing full- shift exposures for each shift for each employee in each job classification in each work area. Representative 30-minute short- term employee exposures shall be determined on the basis of one or more samples representing 30 minute exposures associated with operations that are most likely to produce exposures above the excursion limit for each shift for each job classification in each work area.
(2) Initial monitoring.
(A) Each employer who has a workplace or work operation covered by this standard, except as provided for in subsections (d)(2)(B) and (d)(2)(C) of this section, shall perform initial monitoring of employees who are, or may reasonably be expected to be exposed to airborne concentrations at or above the TWA permissible exposure limit and/or excursion limit.
(B) Where the employer has monitored after March 31, 1992, for the TWA permissible exposure limit and/or the excursion limit, and the monitoring satisfies all other requirements of this section, the employer may rely on such earlier monitoring results to satisfy the requirements of subsection (d)(2)(A) of this section.
(C) Where the employer has relied upon objective data that demonstrate that asbestos is not capable of being released in airborne concentrations at or above the TWA permissible exposure limit and/or excursion limit under the expected conditions of processing, use, or handling, then no initial monitoring is required.
(3) Monitoring frequency (periodic monitoring) and patterns: After the initial determinations required by subsection (d)(2)(A) of this section, samples shall be of such frequency and pattern as to represent with reasonable accuracy the levels of exposure of the employees. In no case shall sampling be at intervals greater than six months for employees whose exposures may reasonably be foreseen to exceed the TWA permissible exposure limit and/or excursion limit.
(4) Changes in monitoring frequency: If either the initial or the periodic monitoring required by subsection (d)(2) and (d)(3) of this section statistically indicates that employee exposures are below the TWA permissible exposure limit and/or excursion limit, the employer may discontinue the monitoring for those employees whose exposures are represented by such monitoring.
(5) Additional monitoring: Notwithstanding the provisions of subsections (d)(2)(B) and (d)(4) of this section, the employer shall institute the exposure monitoring required under subsections (d)(2)(A) and (d)(3) of this section whenever there has been a change in the production, process, control equipment, personnel or work practices that may result in new or additional exposures above the TWA permissible exposure limit and/or excursion limit or when the employer has any reason to suspect that a change may result in new or additional exposures above the PEL and/or excursion limit.
(6) Method of monitoring.
(A) All samples taken to satisfy the monitoring requirements of subsection (d) of this section shall be personal samples collected following the procedures specified in Appendix A.
(B) All samples taken to satisfy the monitoring requirements of subsection (d) of this section shall be evaluated using the OSHA Reference Method (ORM) specified in Appendix A of this section, or an equivalent counting method.
(C) If an equivalent method to the ORM is used, the employer shall ensure that the method meets the following criteria:
1. Replicate exposure data used to establish equivalency are collected in side-by-side field and laboratory comparisons; and
2. The comparison indicates that 90% of the samples collected in the range 0.5 to 2.0 times the permissible limit have an accuracy range of plus or minus 25 percent of the ORM results at a 95% confidence level as demonstrated by a statistically valid protocol; and
3. The equivalent method is documented and the results of the comparison testing are maintained.

(D) To satisfy the monitoring requirements of subsection (d) of this section, employers must use the results of monitoring analysis performed by laboratories which have instituted quality assurance programs that include the elements as prescribed in Appendix A of this section.
(7) Employee notification of monitoring results.
(A) The employer shall, within 15 working days after the receipt of the results of any monitoring performed under the standard, notify the affected employees of these results in writing either individually or by posting of results in an appropriate location that is accessible to affected employees.
(B) The written notification required by subsection (d)(7)(A) of this section shall contain the corrective action being taken by the employer to reduce employee exposure to or below the TWA and/or excursion limit, wherever monitoring results indicated that the TWA and/or excursion limit had been exceeded.
(e) Regulated Areas -
(1) Establishment: The employer shall establish regulated areas wherever airborne concentrations of asbestos and/or PACM are in excess of the TWA and/or excursion limit prescribed in subsection (c) of this section.
(2) Demarcation: Regulated areas shall be demarcated from the rest of the workplace in any manner that minimizes the number of persons who will be exposed to asbestos.
(3) Access: Access to regulated areas shall be limited to authorized persons or to persons authorized by the Chief or the Director.
(4) Provision of respirators: Each person entering a regulated area shall be supplied with and required to use a respirator, selected in accordance with subsection (g)(2) of this section.
(5) Prohibited activities: The employer shall ensure that employees do not eat, drink, smoke, chew tobacco or gum, or apply cosmetics in the regulated areas.
(f) Methods of compliance -
(1) Engineering controls and work practices.

(A) The employer shall institute engineering controls and practices to reduce and maintain employee exposure to or below the TWA and/or excursion limit prescribed in subsection (c) of this section, except to the extent that such controls are not feasible.
(B) Wherever the feasible engineering controls and work practices that can be instituted are not sufficient to reduce employee exposure to or below the TWA and/or excursion limit prescribed in subsection (c) of this section, the employer shall use them to reduce employee exposure to the lowest levels achievable by these controls and shall supplement them by the use of respiratory protection that complies with the requirements of subsection (g) of this section.
(C) Particular tools: All hand-operated and power-operated tools which would produce or release fibers of asbestos, such as, but not limited to, saws, scorers, abrasive wheels, and drills, shall be provided with local exhaust ventilation systems which comply with subsection (f)(1)(E) of this section.
(D) For the following operations, wherever feasible engineering controls and work practices that can be instituted are not sufficient to reduce the employee exposure to or below the TWA and/or excursion limit prescribed in subsection (c) of this section, the employer shall use them to reduce employee exposure to or below 0.5 fiber per cubic centimeter of air (as an eight-hour time-weighted average) or 2.5 fibers/cc for 30 minutes (short-term exposure) and shall supplement them by the use of any combination of respiratory protection that complies with the requirements of subsection (g) of this section, work practices and feasible engineering controls that will reduce employee exposure to or below the TWA and to or below the excursion limit permissible prescribed in subsection (c) of this section:
Coupling cutoff in primary asbestos cement pipe manufacturing; sanding in primary and secondary asbestos cement sheet manufacturing; grinding in primary and secondary friction product manufacturing; carding and spinning in dry textile processes; and grinding and sanding in primary plastics manufacturing.
(E) Local exhaust ventilation: Local exhaust ventilation and dust collection systems shall be designed, constructed, installed, and maintained in accordance with good practices such as those found in the American National Standard Fundamentals Governing the Design and Operation of Local Exhaust Systems, ANSI Z9.2-1979.
(F) Wet methods: Insofar as practicable, asbestos shall be handled, mixed, applied, removed, cut, scored, or otherwise worked in a wet state sufficient to prevent the emission of airborne fibers so as to expose employees to levels in excess of the TWA and/or excursion limit, prescribed in subsection (c) of this section, unless the usefulness of the product would be diminished thereby.
(G) Particular products and operations: No asbestos cement, mortar, coating, grout, plaster, or similar material containing asbestos, shall be removed from bags, cartons, or other containers in which they are shipped, without being either wetted, or enclosed, or ventilated so as to prevent effectively the release of airborne fibers.
(H) Compressed air: Compressed air shall not be used to remove asbestos or materials containing asbestos unless the compressed air is used in conjunction with a ventilation system which effectively captures the dust cloud created by the compressed air.
(I) Flooring: Sanding of asbestos-containing flooring material is prohibited.
(J) The spraying of any substance containing any amount of asbestos in or upon a building or other structure during its construction, alteration, or repair is prohibited. Exceptions: 1. Exterior and interior coating and laminating resins containing encapsulated asbestos fibers bound within the finished product from manufacture through application. 2. Cold process asphalt roof coatings. 3. Substances containing less than one-quarter of one percent asbestos solely as a result of naturally occurring impurities in the substance or its components.
(2) Compliance program.
(A) Where the TWA and/or excursion limit is exceeded, the employer shall establish and implement a written program to reduce employee exposure to or below the TWA and to or below the excursion limit by means of engineering and work practice controls as required by subsection (f)(1) of this section, and by the use of respiratory protection where required or permitted under this section.
(B) Such programs shall be reviewed and updated as necessary to reflect significant changes in the status of the employer's compliance program.
(C) Written programs shall be submitted upon request for examination and copying to the Chief, the Director, affected employees and designated employee representatives.

(D) The employer shall not use employee rotation as a means of compliance with the TWA and/or excursion limit.
(3) Specific compliance methods for brake and clutch repair.
(A) Engineering controls and work practices for brake and clutch repair and service: During automotive brake and clutch inspection, disassembly, repair and assembly operations, the employer shall institute engineering controls and work practices to reduce employee exposure to materials containing asbestos using the negative pressure enclosure/HEPA vacuum system requirements set out in Appendix F to this section. The employer may also comply using an equivalent method which follows written procedures which the employer demonstrates can achieve results equivalent to Method A in Appendix F to this section. For facilities in which no more than 5 pair of brakes or 5 clutches are inspected, disassembled, repaired, or assembled per week, the method set forth in subsection [D] of Appendix F to this section may be used.
(B) The employer may also comply by using an equivalent method which follows written procedures, which the employer demonstrates can achieve equivalent exposure reductions as do the two "preferred methods." Such demonstration must include monitoring data conducted under workplace conditions closely resembling the process, type of asbestos containing materials, control method, work practices and environmental conditions which the equivalent method will be used, or objective data, which document that under all reasonably foreseeable conditions of brake and clutch repair applications, the method results in exposures which are equivalent to the methods set out in Appendix F to this section.
(g) Respiratory protection -
(1) General: For employees who are required to use respirators by this section, the employer must provide respirators that comply with the requirements of this subsection. Respirators must be used during:
(A) Periods necessary to install or implement feasible engineering and work practice controls;
(B) Work operations, such as maintenance and repair activities, or other activities for which engineering and work practice controls are not feasible;
(C) Work operations for which feasible engineering and work practice controls are not yet sufficient to reduce exposure to or below the TWA and/or excursion limit; and
(D) Emergencies.
(2) Respirator program.
(A) The employer must implement a respirator program in accordance with section 5144(b) through (d) (except (d)(1)(C)), and (f) through (m).
(B) The employer must provide a tight-fitting powered, air-purifying respirator instead of any negative pressure respirator specified in Table 1 when:
1. An employee chooses to use this type of respirator; and
2. This respirator provides adequate protection to the employee.
(C) No employee must be assigned to tasks requiring the use of respirators if, based on their most recent examination, an examining physician determines that the employee will be unable to function normally using a respirator, or that the safety or health of the employee or other employees will be impaired by the use of a respirator. Such employees must be assigned to another job or given the opportunity to transfer to a different position, the duties of which they can perform. If such a transfer position is available, the position must be with the same employer, in the same geographical area, and with the same seniority, status, and rate of pay the employee had just prior to such transfer.
(3) Respirator selection.
(A) The employer shall select the appropriate respirator as specified in Table 1.
Table 1. Respiratory Protection for Asbestos Fibers


Airborne concentration of
asbestos or conditions of use. Required respirator
Not in excess of 1 f/cc Half-mask air purifying respirator
(10 X PEL). other than a disposable
respirator,equipped (continued)