ESA BIOSCIENCES INC. VITAMIN D HPLC TEST

K072536 · Esa Biosciences, Inc. · MRG · May 7, 2008 · Clinical Chemistry

Device Facts

• Record ID: K072536
• Device Name: ESA BIOSCIENCES INC. VITAMIN D HPLC TEST
• Applicant: Esa Biosciences, Inc.
• Product Code: MRG · Clinical Chemistry
• Decision Date: May 7, 2008
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1825
• Device Class: Class 2

Intended Use

The ESA Biosciences Inc. Vitamin D HPLC test is for the quantitative determination of 25-hydroxyvitamin D in human serum or EDTA-plasma to be used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions in an adult population.

Device Story

The Vitamin D HPLC Test is a quantitative assay for 25-hydroxyvitamin D in human serum or EDTA-plasma. The system utilizes high performance liquid chromatography (HPLC) with electrochemical (EC) detection. Input samples undergo pre-analytical processing: mixing with precipitation reagent containing an internal standard, centrifugation, and solid phase extraction (SPE) to isolate 25(OH)D. The prepared sample is analyzed via HPLC using a dual coulometric EC cell; the first cell oxidatively screens interfering components, while the second cell is optimized for selective 25(OH)D detection. The device is used in clinical laboratory settings by trained personnel. Output is a quantitative concentration value used by clinicians alongside other laboratory data to assess vitamin D sufficiency and guide patient management. The internal standard corrects for extraction recovery and volume variability. The device provides a standardized method for vitamin D measurement, aiding in the diagnosis and monitoring of vitamin D status in adults.

Clinical Evidence

Bench testing only. Precision studies (CLSI EP5-A) demonstrated total CVs of 5.43%–12.6%. Linearity (CLSI EP6-A) confirmed a measuring range of 7–200 ng/mL (r² > 0.99). Method comparison (CLSI EP9-A2) against a reference HPLC method (n=95) showed a correlation of r=0.973. Limit of Quantitation (LoQ) was 7.0 ng/mL. Interference testing (CLSI EP7-A2) identified 25(OH)EpiD3 as an interferent in infants, leading to the age-based contraindication.

Technological Characteristics

Quantitative HPLC assay with electrochemical (EC) detection. Components include SPE columns, mobile phase, and reagents. Requires ESA HPLC system with CouloChem III or CoulArray EC detector. Dual coulometric cell configuration for interference screening and analyte detection. Single-point internal standard quantitation. Calibrated via UV absorbance spectrometry against NIST SRM 935a.

Indications for Use

Indicated for quantitative determination of 25-hydroxyvitamin D in human serum or EDTA-plasma to assess vitamin D sufficiency in adults. Contraindicated for patients < 1 year of age due to potential interference from C3 epimers.

Regulatory Classification

Identification

A vitamin D test system is a device intended for use in clinical laboratories for the quantitative determination of 25-hydroxyvitamin D (25-OH-D) and other hydroxylated metabolites of vitamin D in serum or plasma to be used in the assessment of vitamin D sufficiency.

Special Controls

*Classification.* Class II (special controls). Vitamin D test systems must comply with the following special controls:(1) Labeling in conformance with 21 CFR 809.10 and (2) Compliance with existing standards of the National Committee on Clinical Laboratory Standards.

Predicate Devices

• LIASON® 25 OH Vitamin D Assay (K032844)

Submission Summary (Full Text)

{0}------------------------------------------------

Ko7253 6

MAY - 7 2008

# 510(k) SUMMARY

This summary of 510(k) Safety and effectiveness information is being submitted in
accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is:

| Submitter: | ESA Biosciences Inc.<br>22 Alpha Road<br>Chelmsford, MA 01824<br>USA<br>Phone: [phone]<br>Fax: [phone] |
|---------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Contact Person: | Harold Asp<br>Quality Assurance Manager |
| Date of Summary<br>Preparation: | May 05, 2008 |
| Device Name: | ESA Biosciences Inc. Vitamin D HPLC Test |
| Classification Name: | Vitamin D Test System 21CFR 862.1825 Product Code:<br>MRG |
| Predicate Device: | LIASON® 25 OH Vitamin D Assay<br>510(k) 032844 |
| Statement of<br>Intended Use: | The ESA Biosciences Inc. Vitamin D HPLC test is for the<br>quantitative determination of 25-hydroxyvitamin D in<br>human serum or EDTA-plasma to be used in the<br>assessment of vitamin D sufficiency. Assay results<br>should be used in conjunction with other clinical or<br>laboratory data to assist the clinician in making individual<br>patient management decisions in an adult population. |

{1}------------------------------------------------

# Device Description

The ESA method is a complete kit for measurement of Total 25(OH)D by HPLC with electrochemical (EC) detection. Specific reagents and solid phase extraction columns are included for sample preparation and are employed with user-supplied standard laboratory equipment (centrifuge, test tubes, pipettes, etc.). A 200µL volume of sample (serum or plasma) is mixed with a precipitation reagent, which contains internal standard (IS).

The internal standard is a stable vitamin D analogue that is used to correct for variability in extraction recovery and analytical sample volume. After centrifugation, supernatant is poured onto a pre-conditioned SPE column for rapid extraction of 25(OH)D and IS. SPE columns are washed with 2 different reagents and analytes are eluted with a third reagent. The resulting eluent is diluted before analysis. The prepared sample is analyzed with an isocratic HPLC system using an ESA EC detector (Coulochem® III or CoulArray®) equipped with a dual coulometric EC cell. Calibration is accomplished by direct HPLC analysis of authentic standard solutions (i.e. not taken through the extraction step). Analysis requires a specific guard and analytical column, mobile phase and calibration reagents to allow rapid quantitative analysis.

A dual EC cell is used with the first, upstream, cell maintained at a specific potential to oxidatively screen possible interfering sample components. The second, downstream cell is maintained at a potential that is optimized for selective 25(OH)D detection. The dual coulometric EC cell is a rugged detector that provides much higher selectivity than commonly used absorbance detectors. This allows the use of lower sample volumes than are typically required with HPLC-UV methods and is less susceptible to interferences. Analytical run time is less than 12 minutes and Total 25(OH)D sample concentration is automatically determined by single-point internal standard quantitation.

# Summary of Performance

## Precision

Four 'neat' (i.e., not spiked or pooled) samples consisting of 2 sera and 2 EDTA plasma were used for this study, which followed CLSI EP5-A. Four replicates of each sample, were individually prepared (taken through all pre-analytical steps of the procedure) and analyzed in a single run each day, repeated over 20 days. Samples were run on a single instrument, by two operators and using two lots of reagents and extraction columns. Analysis of variance calculations were used to estimate within-run and within-device imprecision.

| Assay | Mean | Within-run | | Within-device | | N |
|--------------------|-------|------------|------|---------------|------|----|
| | ng/mL | SD (ng/mL) | % CV | SD (ng/mL) | % CV | |
| Total 25(OH)D | | | | | | |
| Sample #1 (Plasma) | 22.1 | 0.63 | 2.82 | 1.34 | 6.05 | 79 |
| Sample #2 (Plasma) | 21.8 | 0.50 | 2.31 | 1.19 | 5.48 | 80 |
| Sample #3 (Serum) | 25.5 | 0.65 | 2.55 | 1.44 | 5.66 | 80 |
| Sample #4 (Serum) | 25.6 | 0.55 | 2.16 | 1.39 | 5.43 | 78 |

#### Precision Performance

Precision performance was also evaluated using plasma pools supplemented with 25(OH)D at 2 concentration levels. The protocol followed CLSI EP5-A. Five replicates of each level were individually prepared (i.e., taken through all pre-analytical steps) and analyzed in a single run each day. Data from a total of 20 runs, performed at two sites were included in the

{2}------------------------------------------------

precision evaluation (300 total observations). Three different HPLC-EC systems, four operators, two reagent lots, five analytical columns (3 lots), three lots of SPE columns and four analytical cells were used in these studies. Analysis of variance calculations were used to estimate withinrun and total imprecision.

| Assay | Mean | Within-run | | Total | | N |
|---------------|-------|------------|------|------------|------|-----|
| | ng/mL | SD (ng/mL) | % CV | SD (ng/mL) | % CV | |
| Total 25(OH)D | | | | | | |
| Level 1 | 43.8 | 4.6 | 10.6 | 5.5 | 12.6 | 100 |
| Level 2 | 117.2 | 8.0 | 6.8 | 9.9 | 8.4 | 100 |

#### Precision Performance

## Limit of Detection (LoD)

The LoD was determined consistent with the guidelines in CLSI EP17A. Blank matrix was bovine serum albumin in phosphate buffered saline. Patient samples with low 25(OH)D concentrations, as determined by the LCUV reference method, were used to prepare low level pools The results were analyzed using the nonparametric method. The LoQ (limit of quantitation) was set to the concentration at which the % CV from low level samples was ≤ 20%.

Provided below are estimates of LoD, limit of blank (LoB) and LoQ based on 133 determinations in 11 runs, with 67 blank and 66 low-level samples with less than 5% false negative and less than 5% false positives:

| | ng/mL | | | |
|---------------|-------|-----|-----|-----|
| Assay | LoB | LoD | LoQ | N |
| Total 25(OH)D | 2.5 | 5.0 | 7.0 | 133 |

#### LoD. LoB. and LoQ for Vitamin D

#### Linearity

Linearity was studied according to CLSI EP6 on two HPLC systems. Low and high level plasma were mixed proportionally to obtain a total of 13 equally -spaced concentrations spanning the intended assay range (7.0 - 200 ng/mL) for 25(OH)D. Three aliquots of each sample concentration were taken through all pre-analytical steps of the assay and analyzed in random order in a single run. Performance was assessed using the polynomial evaluation as described in CLSI EP-6A.

For total 25(OH)D by the ESA Vitamin D HPLC Test, the method has been demonstrated to be linear from the LoQ (7.0 ng/mL) to 200 ng/mL based on goals of ≤ 3ng/mL bias due to nonlinearity for concentrations of ≤ 20 ng/mL and ≤ 7.5% bias due to non-linearity at concentrations of 21-200 ng/mL.

{3}------------------------------------------------

### Recovery

Sample pools: EDTA plasma (5 subjects) and serum (6 subjects) were each spiked with 25HOD concentrate to increase the individual concentrations of 25(OH)D by 0, 5, 10, 20, 50, 100 and 200 ng/mL. Concentration levels of total 25(OH)D in unspiked pools were less than 10 ng/mL. Three replicates of each sample were taken through all pre-analytical steps of the assay. Each pool and corresponding spiked samples were analyzed in random order in a single run. Samples were analyzed on two different HPLC systems.

Percent recovery was determined by: [(Measured concentration - Endogenous (unspiked) concentration)/Spiked Concentration] x 100.

For each sample matrix, mean recovery (n = 6, each level = 3 replicates on 2 systems) are summarized below:

| Sample | Spiked Concentration<br>(ng/mL) | % Recovery Mean<br>(n=6) | Standard Deviation<br>% (n=6) |
|--------|---------------------------------|--------------------------|-------------------------------|
| Plasma | 10 | 90 | 20 |
| | 20 | 100 | 10 |
| | 40 | 100 | 11 |
| | 100 | 99 | 6.8 |
| | 200 | 107 | 7.2 |
| Serum | 10 | 93 | 20 |
| | 20 | 94 | 13 |
| | 40 | 96 | 8.9 |
| | 100 | 96 | 5.5 |
| | 200 | 105 | 3.8 |

#### Recovery 25(OH)D

### Method Comparison and Bias Estimation

Method comparison studies followed CLSI EP9-A2. The studies were performed at two external sites. Serum samples from 85 individual patients were collected at the University of Wisconsin Hospitals and Clinics (UWHC). These were chosen from samples submitted routinely to UWHC for 25(OH)D analysis and represent a wide population with respect to medical, dietary and therapeutic conditions. Fifteen additional samples were prepared by augmenting aliquots of a single serum pool (prepared from approximately 50 individual patient samples) with varying amounts of 25(OH)D.

All 100 samples were run in duplicate by a LCUV reference method at UWHC. Fifty of these patient samples were run in duplicate by the ESA LCEC test method at one external site and the other fifty patient samples were run in duplicate by the LCEC test method at UWHC. Samples were run on 12 separate days, by 3 different technologists over a 7 week period with no more than 10 patient samples run per day. Calibration and control materials used for the LCUV reference method were independent of the LCEC test method.

For this analysis, data from both sites were pooled and the first replicate of the Y (ESA LCEC) results were analyzed against the average of the X (LCUV) results. No outliers were removed. Five samples with values outside the measuring range, LOQ (7.0 ng/mL) to 200 ng/mL, of the ESA assay were excluded from this analysis.

{4}------------------------------------------------

The following table and figure provide a summary of data analysis for total 25(OH)D. All data analyses were performed as described in EP9-A2 and include the following:

- A Number of points used in the regression and range of X data.
- Slope and intercept of fitted ordinary least squares and Deming regression lines. ♪
- A From least squares regression: correlation coefficient, standard error of estimate (calculated in the vertical (Y) direction) and confidence intervals for the slope and intercept.
- Scatter plots with the line of identity. A
- Predicted bias and confidence intervals calculated at various X values from the A partitioned bias method.

| 25(OH)D | |
|--------------------------------------------|--------------------------|
| Equation: Least Squares | LCEC = 1.027(LCUV) - 5.3 |
| Slope, 95% Confidence Interval | 0.976 to 1.077 |
| Intercept, 95% Confidence Interval (ng/mL) | -8.7 to -1.8 |
| Correlation Coefficient, R | 0.973 |
| N | 95 |
| Standard Error (ng/mL, y-direction) | 9.91 |
| Range (ng/mL) | 9.6 – 195 |
| Equation: Deming | LCEC = 1.056(LCUV) - 6.9 |

#### Regression Summary -- Total 25(OH)D

{5}------------------------------------------------

Image /page/5/Figure/0 description: The image is a scatter plot titled "Total 25(OH)D, LCEC vs. LCUV". The x-axis is labeled "LCUV, ng/mL" and ranges from 0 to 200. The y-axis is labeled "LCEC, ng/mL" and ranges from 0 to 200. The plot shows a series of data points labeled "Series 1" and a line labeled "Identity".

Total 25(OH)D - Scatter Plot of First LCEC Replicate vs. Average of Reference Method with Line of Identity

Total 25(OH)D First LCEC Rep vs. Avg. LCUV

Because the correlation coefficient (r) fails the adequate range test (i.e., r < 0.975), the partitioned bias method was used.

| | Total 25(OH)D - Bias and Confidence Intervals Using Partitioned Bias Method |
|--|-----------------------------------------------------------------------------|
|--|-----------------------------------------------------------------------------|

| Average LCUV<br>(ng/mL) | Range of<br>LCUV<br>(ng/mL) | N | Bias<br>(ng/mL) | Bias<br>(%) | Lower 95%<br>CI Bias*<br>(ng/mL) | Upper 95%<br>CI Bias*<br>(ng/mL) |
|-------------------------|-----------------------------|----|-----------------|-------------|----------------------------------|----------------------------------|
| 22.9 | 9.6 – 34.4 | 32 | -2.6 | -11.2 | -17.4 | -4.9 |
| 45.4 | 34.5 – 56.1 | 31 | -5.6 | -12.3 | -18.4 | -6.1 |
| 100.3 | 57.0 - 195 | 32 | -3.3 | -3.3 | -8.5 | 2.0 |

* Confidence intervals calculated at the indicated average LCUV concentration

{6}------------------------------------------------

## Interference Studies

This assay is susceptible to interference from 25(OH)epiD2 and 25(OH)epiD3, which are endogenous metabolites present in neonates . This assay should therefore not be used for measurement in samples from patients < 1 year of age. A 10% positive bias was observed for samples with a cholesterol level of 500mg/dL. Estimated bias for lower concentrations of cholesterol was acceptable (<10%). The following provides additional detail of interference susceptibility testing.

Interference susceptibility testing was performed according to CLSI EP7-A. Substances chosen for study were based on consideration of chemical structure determinants for electrochemical response and chromatographic retention, common medications in the intended population, therapeutic vitamin D analogs and matrix effects / conditions. The following table lists substances that were initially screened by direct HPLC-EC analysis along with the observed retention times.

| Compound / Class | Alternate and Commercial<br>Names | Retention time (min.) for<br>peaks observed on<br>analytical channel |
|--------------------------------------------|-----------------------------------|----------------------------------------------------------------------|
| <i>Lipid-soluble vitamins</i> | | |
| Retinyl Acetate | | 25.06 |
| Retinol | | 11.5 |
| α-Tocopherol | | None |
| Coenzyme Q10 | | None |
| β-Carotene | | None |
| Lutein | | None |
| γ-Tocopherol | | None |
| δ-Tocopherol | | None |
| Lycopene | | None |
| <i>Vitamin D Compounds /<br/>Analogues</i> | | |
| D3 | Cholicalciferol | None |
| D2 | Ergocalciferol | None |
| 1α,25(OH)2D3 | Calcitriol, Rocaltrol, Calcijex | 4.08 |
| 19-nor-1α,25(OH)2D2 | Paricalcitol, Zemplar | 3.74 |
| Dihydrotachysterol | | None |
| 1α,25(OH)2D2 | | 4.7 |
| 1α(OH)D3 | One-alpha, Alfarol | 33.9 |
| 1α(OH)D2 | Doxercalciferol, Hectorol | 34 |
| 25(OH)epiD3 | | 8.46 |

#### Compounds Analyzed by Direct HPLC Analysis

From Table 9-1, 19-nor-1α,25(OH)-D2 (paricalcitol) and 25(OH)epiD3 were the only compounds that were identified as possible interfering substances. These substances and several endogenous compounds were characterized to determine the degree of interference as a function of interferent concentration (i.e., dose-response). Serum was prepared with high and low (normal) concentrations of each possible interferent (see below). High and low samples were mixed proportionally to produce a total of 5 equally-spaced levels of each possible interfering substance. Three replicates of each sample were individually prepared and analyzed in random order a single run.

{7}------------------------------------------------

The results of these studies are described below:

The following endogenous substances, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, did not interfere at, or below, the interferent test concentration indicated. Bias, calculated from regression analysis of the doseresponse data, exceeding 10% is considered interference.

| Substance | High Test<br>Concentration | Comments |
|---------------|----------------------------|------------------------|
| Triglycerides | 1571 mg/dL | Test Material Triolein |
| Albumin | 12.7 g/dL | |
| Bilirubin | 60 mg/dL | . |
| Hemoglobin | 500 mg/dL | Gross hemolysis |

The following vitamin D analog, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, did not interfere at the interferent test concentration indicated. Bias, calculated from regression analysis of the dose-response data, exceeding 10% is considered interference.

| Substance | High Test<br>Concentration | Comments |
|---------------|----------------------------|----------------------------------------------------------|
| Paricalcitrol | 100 ng/mL | Approx 100 times therapeutic<br>range for this compound. |

The following endogenous substance, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL was found to produce a positive bias of 10% for both analytes at the high test concentration indicated. Acceptable bias (i.e., < 10%) was found for interferent concentrations < 375mg/dL.

| Substance | High Test<br>Concentration |
|-------------|----------------------------|
| Cholesterol | 500 mg/dL |

The C3 epimer of 25(OH)D, 25(OH)EpiD3, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, was found to interfere with 25(OH)D. As reported by Singh et al.", 25(OH)D2 or D3 epimers may account for a significant proportion (8.7-61.1%) of the total 25(OH)D in infants, with concentrations ranging from 5-92ng/mL. No C3 epimers were detected in age groups greater than 0-1 year. Based on this report, this assay should not be susceptible to interference from C3 epimers for the intended adult patient population.

{8}------------------------------------------------

## Reference Range

The following results were obtained specifically with the ESA Vitamin D HPLC Test from a reference sample group that is reflective of a typical sample population within the contiguous 48 US States. Note: These data are provided as an example. It is important for each laboratory to establish a reference range representative of its typical population.

Samples from 150 apparently healthy adult human subjects were collected from 3 locations to achieve even North-South geographical distribution within the latitudes of the contiguous 48 US States. Samples were collected in late winter. Individuals taking prescription or over-the-counter vitamin D supplements were excluded. The reference range was determined according to the non-parametric method in CLSI C28-A2.

## Observed Reference Range and Frequency Distribution

| Population (N) | Median 25(OH)D ng/mL | Observed Range ng/mL<br>2.5th to 97.5th Percentile |
|----------------|----------------------|----------------------------------------------------|
| 150 | 17.4 | 5.7 - 34.8 |

Image /page/8/Figure/5 description: The image is a bar graph showing the frequency (%) of a population across different concentration ranges (ng/mL). The x-axis represents the concentration range, while the y-axis represents the frequency percentage. The concentration ranges include <5.0, 5.1-10.0, 10.1-15.0, 15.1-20.0, 20.1-25.0, 25.1-30.0, 30.1-35.0, and 35.1-40.0. The highest frequency is observed in the 15.1-20.0 range, with approximately 25% of the population.

#### 25(OH)D Reference Sample Distribution

{9}------------------------------------------------

| Similarities | | |
|------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Item | Device | Predicate |
| Intended Use | For the quantitative<br>determination of 25-<br>hydroxyvitamin D in human<br>serum or EDTA-plasma to be<br>used in the assessment of<br>vitamin D sufficiency. Assay<br>results should be used in<br>conjunction with other clinical<br>or laboratory data to assist the<br>clinician in making individual<br>patient management decisions in<br>an adult population. | For in vitro diagnostic use.<br>Quantitative determination of<br>25-Hydroxyvitamin D (25-<br>OH-D) and other hydroxylated<br>vitamin D metabolites in<br>human serum or plasma to be<br>used in the assessment of<br>vitamin D sufficiency. Assay<br>results should be used in<br>conjunction with other clinical<br>laboratory data to assist the<br>clinician in making patient<br>management decisions in an<br>adult population. |
| Indications for<br>Use | Determination of Vitamin D<br>sufficiency. | Same |
| Matrix | Human serum or plasma | Same |
| Differences | | |
| Item | Device | Predicate |
| Analyte | 25-Hydroxyvitamin D | Same |
| Methodology | High Performance Liquid<br>Chromatography with<br>Electro-Chemical Detection | Chemiluminescent immunoassay |
| Kit Controls | Recommend commercially<br>available controls | Two levels, horse serum based,<br>extracted identically to patient<br>samples |
| Standards | Working single point<br>calibrator is an aqueous<br>calibration stock solution and<br>internal stock solution | Stored Master Curve based on 10<br>points, derived from serum based<br>standards extracted identically to<br>controls and patient samples |

# Technical Characteristics Compared to Predicate:

# Conclusion

Through the use of plasma and serum samples, augmented plasma and serum samples, standards and controls the performance and reliability of this assay has been validated over the measuring range of 7.0 ng/mL (LoQ) to 200 ng/mL. In so doing adequate over the modeling fungs and imits and immits and immunity from interferences has been demonstrated.

{10}------------------------------------------------

# Standards Referenced

- EP9-A2 Method Comparison and Bias Estimation Using Patient Samples: Approved 1. Guideline- Second Edition
- C28-A2 How to define and Determine Reference Intervals in the Clinical Laboratory; 2. Approved Guideline-Second Addition
- EP6-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical 3. Approach; Approved Guideline
- EP7-A Interference testing in Clinical Chemistry; Approved Guideline 4.
- EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; ട്. Approved Guideline-Second edition

# Literature Referenced

- Lensmever GL, Wiebe DA, Binkley N, Drezner MK. HPLC method for 25-hydroxyvitamin 1. D measurement: comparison with contemporary assays. Clin Chem 2006;52:1120-6.
- 2. Singh RJ, Taylor RL, Reddy GS, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab 2006;91:3055-61.

{11}------------------------------------------------

## DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/11/Picture/1 description: The image shows the seal for the Department of Health & Human Services - USA. The seal is circular, with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter of the circle. In the center of the seal is an image of an eagle.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

ESA Biosciences Inc. c/o Mr. Harold Asp Quality Assurance Manager 22 Alpha Road Chelmsford, MA 01824

MAY - 7 2008

Re: k072536

Trade/Device Name: Vitamin D HPLC Test Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D Test system Regulatory Class: Class II Product Code: MRG Dated: March 24, 2008 Received: March 26, 2008

Dear Mr. Asp:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

{12}------------------------------------------------

Page 2 -

This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number [phone] or [phone] or at its Internet address at http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Jean M. Cooper, M.S., D.V.M.

Yean M. Cooper, M.S., D.V.M. Director Division of Chemistry and Toxicology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

{13}------------------------------------------------

# Indication for Use

510(k) Number (if known):

Device Name: ESA Biosciences Inc. Vitamin D HPLC Test

Indication For Use: k072536

The ESA Biosciences Inc. Vitamin D HPLC test is for the quantitative determination of 25-hydroxyvitamin D in human serum or EDTA-plasma to be used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions in an adult population.

Prescription Use _ X (21 CFR Part 801 Subpart D)

And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Di/ Sign. Off.

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) k072536