Review Team (February 2016)

Bio-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Evaluation of Method Performance vs. SMPR requirements.

Method Reference # Method title:

Determination of biotin in infant, pediatric and adult nutritionals by HPLC and fluorescence detection

This reverse phase HPLC method with post column reduction and fluorescence detection allows for the quantitative determination of biotin in infant, pediatric, and adult nutritionals. Sample of appropriate size is mixed with 6% meta-phosphoric acid to precipitate out the protein to produce a filtrate. The filtrate is injected onto a C18 HPLC column where biotin and riboflavin are separated with a mobile phase (20% methanol in 0.02 M phosphate buffer at pH7.0). The biotin after eluting from the column binds with the streptavidin fluorescein to become a fluorescent conjugate. The conjugate is then detected by fluorescence at an excitation wavelength of 495 nm and an emission wavelength of 518 nm.

Principle of the method:

Suitability Ranking (1-3-5) (select from drop- down list, 5 = best)

Method Performance

Please report

SMPR Requirement

Parameter

in units as stated in SMPR!

All SPIFAN matrices analyzed for repeatability. Representative matrices from each category analyzed for recovery during SLV, except for Infant RTF due to the defficient sample amount.

Applicable to all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). Combination of milk, soy, hydrolyzed protein, FOS/GOS, amino acids, with high or low fat.

SPIFAN matrices

All required analytes are measured.

Method measures all required analytes.

Biotin

0.1-150 mcg/100g reconstituted final product

1.66-142 mcg/100g reconstituted final product

Analytical Range

Biotin

Not Applicable

Limit of detection (LOD)

Biotin

~0.8 mcg/100g in reconsitituted powder product OR ~1.5 mcg/100 g in ready-to-feed product See Table 1

d 0.1 mcg/100g reconstituted final product

Limit of detection (LOQ)

0.1-1 mcg/100g reconstituted final product: 80-120% >1 mcg/100g recons tituted final product: 90-110%

Not Applicable

Spike recovery

Accuracy/Recovery

Range: 95-111% See Table 2

See Table 3

Bias vs SRM

Not Applicable

0.1-1 mcg/100g reconstituted final product : d % >1 mcg/100g reconstituted final product : d 6%

Range: 0.5-3.0% See Table 4

Repeatability (RSDr)

No SMRP requirements

Range: 1.3-4.5% See Table 4

Intermediate Reproducibility (RSDiR)

0.1-1 mcg/100g reconstituted final product : d % >1 mcg/100g reconstituted final product : d %

Reproducibility (RSDR)

90-110% recovery of SRM with different sample sizes and being quantified withing the calibration curve range of 8.5-60 ng/mL. See Table 5

Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range.

Adequate proof of performance via system suitability

Feedback from Users of the Method since being awarded First Action Official Methods Status

Did Method Author Consider ERP’s Method Specific Recommendations (See web link to specific method comments):

Analytical equipment is commonly available in most labs.

Analytical equipment

Proprietary equipment

No unique proprietary equipment/accessories are required.

hod does not require any special safety precautions e.g. personal protection from highly toxic solve

Laboratory safety

Other Considerations

0

Overall Score

Decision by ERP Recommendation ERP 2 years after First Action Status

move to Final Action/repeal/remove/expand 2 year term

Notes a Concentrations apply to (1)

"ready to feed liquids" "as-is"; (2) reconstituted powders (25 g into 200 g water); and (3) liquid concentrates diluted 1:1 by weight.

b Units

SPIFAN ERP Checklist v 1.0