3-Fold Faster Polybrominated Diphenyl Ether (PBDE) - Cp-Analytica

Global Restek Advantage 

2009.03 

Achieve Faster Analyses on Any 

HPLC System Using Ultra II 

Columns 

• Designed for scalability and optimal 

chromatography on any LC system. 

• Comprehensive range of particle 

sizes—1.9µm, 2.2µm, 3µm, and 5µm. 

• Available in traditional phases and 

unique chemistries for alternate 

selectivity. 

Ultra High Pressure Liquid Chromatography (UHPLC) is 

arguably the most significant recent advancement in liquid chro- 

matography. In the past few years, we have experienced an evolu- 

tionary leap in system performance that has altered our analysis time expectations for liquid sep- 

arations. Implementing UHPLC technology can certainly produce faster separations and increase 

laboratory productivity, but adopting the technology requires substantial capital expenditures. 

Significant savings, in both time and solvent usage, are available to most labs right now, without 

the costly upgrade to UHPLC instrumentation and the associated hardware. These savings can be 

realized by evaluating and updating current methodologies that are time- and solvent-consum- 

ing. In this article, we will look at some simple steps and strategic column choices, which can sig- 

nificantly speed up analyses and decrease operating costs on existing systems. 

Chromatography Chromatography Products Products 

www.restek.com 

www.restek.com

Contents 

Pharmaceutical 

Re-Developing your Current Impurity Assay 

for High Sample Throughput ......................1 

Petrochemical 

Fully Resolve C1-C5 Impurities in Ethylene 

According to ASTM D6159................................ 4 

Foods, Flavors & Fragrances 

Meet New Requirements for Melamine 

Analysis at 1µg/g in Infant Formula.............6 

Environmental 

3-Fold Faster Polybrominated Diphenyl 

Ether (PBDE) Using Your Existing 

Instrumentation............................................8 

Clinical/Forensic/Toxicology 

5 Minute Analysis of Vitamin D in Serum 

by LC/MS/MS . . . ............................................ . 10 

Patents & Trademarks 

Restek patents and trademarks are the property of 

Restek Corporation. Other trademarks appearing in 

Restek literature or on its website are the property of 

their respective owners. 

CP-ANALYTICA GmbH 

Am Pulverturm 17 

A-2130 Mistelbach 

tel +43 (0)2572/4381 

fax +43 (0)2572/20791 

info@cp-analytica.at 

www.cp-analytica.at 


Re-Developing your Current Impurity 

Assay for High Sample Throughput 

Step 1: Evaluate Selectivity for the Greatest Speed and Savings 

Although we often look exclusively to UHPLC for speed, by first utilizing column selectivi- 

ty, many time-consuming methods can be updated to show immediate savings. For exam- 

ple, a common assay for famotidine and associated impurities can be improved by using 

new column chemistries. The traditional USP method calls for a high concentration, citrate 

buffered aqueous phase, an acetonitrile organic mobile phase, a traditional analytical scale 

C18 column (250mm x 4.6mm, 5µm), and a 45 minute gradient, resulting in a relatively 

slow analysis that requires a large solvent volume (Figure 1). 

Figure 1 The conventional USP assay for famotidine and associated impurities is 

slow and expensive in terms of time and solvent use. 

Peak List Ret. Time (min.) 

1. impurity A 2.833 

2. impurity B 5.462 

3. impurity C 7.905 

4. famotidine 10.062 

5. impurity D 11.874 

LC_PH0496 

Sample: Conditions: 

Inj.: 10µL Instrument: Shimadzu Prominence UFLCXR 

Conc.: 100µg/mL famotidine, 10µg/mL each impurity Mobile phase: A:100mM sodium citrate trihydrate in 

Sample diluent: methanol water (pH 6.0):acetonitrile (93:7) 

B:acetonitrile 

Column: Allure ® C18 Time (min.) %B 

Cat.#: 9164575 0 0 

Dimensions: 250mm x 4.6mm 15 0 

Particle size: 5µm 42 48 

Pore size: 60Å 43 0 

45 0 

Flow: 1.5mL/min. 

Temp.: 35°C 

Det.: UV @ 268nm 

Figure 2 Switch to a more retentive and selective Ultra II Aromax column and 

reduce analysis time by ~70% and solvent volume by ~60%. 

Fast! 5µm 

Sample: 

Inj.: 10µL 

Conc.: 100µg/mL famotidine, 10µg/mL each impurity 

Sample diluent: methanol 

Column: Ultra II Aromax 

Cat.#: 9607565 

Dimensions: 150mm x 4.6mm 

Particle size: 5µm 

Pore size: 100Å 

• 2 • 


1. impurity A 

2.famotidine 

3. impurity D 

4. impurity C 

5. impurity B 

Conditions: 

Instrument: 

Mobile phase: 

Flow: 

Temp.: 

Det.: 

LC_PH0495 

3.779 

8.264 

9.180 

9.911 

14.018 

Shimadzu Prominence UFLCXR 

20mM potassium phosphate 

(pH 2.5):methanol 

Time (min.) %B 

0 5 

15 35 

2.0mL/min. 

40°C 

UV @ 268nm 

www.restek.com

Figure 3 Switch to a 3µm Ultra II Aromax column and reduce both 

time and solvent usage—by over 80%. 

Faster! 3µm 

Sample: 

Inj.: 5µL 

Conc.: 100µg/mL famotidine, 10µg/mL each impurity 

Sample diluent: methanol 

Column: Ultra II Aromax 

Cat.#: 9607313 






2. famotidine 4.460 




LC_PH0494 

Conditions: 

Instrument: Shimadzu Prominence UFLCXR 

Mobile phase: 20mM potassium phosphate 

(pH 2.5):methanol 


0 5 

8 35 

Flow: 1.2mL/min. 

Temp.: 40°C 

Det.: UV @ 268nm 

Figure 4 Scale down to a 2.2µm Ultra II Aromax column and cut analy- 

sis time and solvent use—by over 90%—without specialized UHPLC 

equipment. 



Fastest! 2.2µm 2. famotidine 2.082 




LC_PH0493 

To speed up this method and reduce solvent consumption, 

we switched columns from a traditional C18 to a 

Restek Ultra II Aromax column (Figure 2). This novel 

bonded phase is both more retentive and more selective, 

which allows a smaller column length (150mm) 

and a faster gradient profile to be used. Since the selectivity 

of the Ultra II Aromax phase is enhanced with 

a methanolic mobile phase, the organic solvent was 

changed to methanol, which is currently a less expensive 

and more readily available solvent than acetonitrile. 

In addition, the new bonded phase allows the high concentration 

citrate buffer to be replaced with the lower 

concentration phosphate buffer. (Note: Citrate buffer in 

high concentrations has been shown to attack stainless 

steel in an HPLC and requires extensive, time-consuming 

flushing to remove.) By making a strategic column 

choice based only on selectivity, analysis time was 

reduced by nearly 70% and organic solvent use was 

reduced by nearly 60% (Table I, page 11). 

Use the flexibility of Restek’s 

Ultra II columns to trim costs 

and speed up analysis time on 

your current LC system. 

Step 2: Scale Analyses to the Optimal 

Particle Size 

Although choosing a column based on selectivity alone 

significantly improves analysis speed and reduces sol- 

vent costs, this method can be further optimized by 

scaling the analysis to a 3µm particle size (Figure 3). 

Ultra II Aromax columns are designed to be fully 

scalable and are available on a wide range of particle 

sizes to support labs interested in speeding up analysis 

times by switching to smaller diameter particles. While 

the pressure increase seen from this change was approx- 

Sample: Conditions: imately two-fold, it is still well within the limits of what 

Inj.: 2µL Instrument: Shimadzu Prominence UFLCXR 

Conc.: 100µg/mL famotidine, 10µg/mL each impurity Mobile phase: 20mM potassium phosphate 

Sample diluent: methanol (pH 2.5):methanol 


Column: Ultra II Aromax 0 5 

Cat.#: 9607853 4 35 

Dimensions: 50mm x 3.0mm Flow: 1.2mL/min. 

Particle size: 2.2µm Temp.: 40°C 

Pore size: 100Å Det.: UV @ 268nm 

Universal application for Any LC System, providing scalability 

and unsurpassed selectivity on a wide range of particle sizes. 

www.restek.com/ultra2 

Global RESTEK Advantage • 3 • 

a normal HPLC will allow. The time and solvent savings 

due to scaling were both about 80% relative to the orig- 

inal method (Table I). 

Further savings can be realized by dropping the parti- 

cle size to the pressure limit of the LC system and 

mobile phase. In this example we used a Shimadzu 

Prominence UFLCXR, a system capable of 660 bar 

maximum pressure, which allowed us to further scale 

the method to an intermediate 2.2µm particle diame- 

ter (Figure 4). With this system and column configura- 

tion, the analysis time is reduced to 5.5 minutes, 

including equilibration, and the mobile phase con- 

sumption is under 7mL per sample. This adds up to a 

time and solvent volume savings of over 90%—with- 

out any investment in specialized UHPLC equipment 

(Table I). 

Continued on page 11. 

www.restek.com

Petrochemical 

Fully Resolve C1-C5 Impurities in 

Ethylene According to ASTM D6159 

Using New High-Capacity Rt ® -Alumina BOND and Rtx ® -1 GC Columns 

Ethylene is one of the highest volume chemicals pro- 

duced in the world, with global production exceeding 

100 million metric tons annually. Ethylene is primarily 

used in the manufacture of polyethylene, ethylene 

oxide, and ethylene dichloride, as well as many other 

lower volume products. Most of these production 

processes use various catalysts to improve product 

quality and process yield. Impurities in ethylene can 

damage the catalysts, resulting in significant replace- 

ment costs, reduced product quality, process downtime, 

and decreased yield. 

Figure 1 Methane and ethane are well resolved in high purity ethylene 

samples. 

Peak List 

1. methane 

2. ethane 

3. ethylene 

Ethylene is typically manufactured through the use of 

steam cracking. In this process, gaseous or light liquid 

hydrocarbons are combined with steam and heated to 

Sample: ethylene 

750-950°C in a pyrolysis furnace. Numerous free radi- Inj. temp.: 200°C 

cal reactions are initiated and larger hydrocarbons are 

converted (cracked) into smaller hydrocarbons. The 

high temperatures used in steam cracking promote the 

formation of unsaturated or olefinic compounds like 

ethylene. Ethylene feedstocks must be tested to ensure 

that only high purity ethylene is delivered for subse- 

quent chemical processing. 

Testing typically follows ASTM D6159-97, a GC/FID 

method which employs a two-column configuration 

consisting of an alumina PLOT column with KCl deac- 

tivation (50m x 0.53mm ID) coupled to a 100% 

dimethyl polysiloxane column (30m x 0.53mm ID x 

5.0µm df ). 

Samples of high purity ethylene typically contain only 

two minor impurities, methane and ethane, which can 

be detected in low ppmV concentrations (Figure 1). 

However, steam cracking can also produce higher 

molecular weight hydrocarbons, especially when 

propane, butane, or light liquid hydrocarbons are used 

as starting materials. Although fractionation is used in 

the final production stages to produce a high purity 

ethylene product, it is still important to be able to identify 

and quantify any other hydrocarbons present in an 

ethylene sample.Achieving sufficient resolution of all of 

these compounds can be challenging due to their similarities 

in boiling point and chemical structure. ASTM 

D6159-97 addresses this issue by combining the separation 

power of two different types of capillary columns. 

The Rt ® -Alumina BOND/KCl PLOT column has excel- 

lent separation capabilities for low molecular weight 

hydrocarbons ranging from C1 through C12, but com- 

plete resolution of all compounds is not always possi- 

ble, depending on the conditions that are employed. 

Figure 2 shows the analysis of an ethylene sample that 

has been spiked with the typical hydrocarbons that may 

be present after ethylene production. When using the 

temperature conditions supplied in the method, there 

are coelutions between three different peak pairs. 

Acetylene and isobutane (peaks 7 and 8) elute at the 

Global RESTEK Advantage 

Column: Rt ® -Alumina BOND/KCl, 50m, 0.53mm ID, 10.0µm (cat.# 19760) 

in series with Rtx ® -1, 30m, 0.53mm ID, 5.0µm (cat.# 10179), 

connected using a Universal Press-Tight ® Connector (cat.# 20401) 

Inj.: 1µL split, 60mL/min. split vent flow rate 

2mm splitless liner (cat.# 20712) 

Carrier gas: helium, constant pressure (8.0psi, 55.2kPa) 

Linear velocity: 25.4cm/sec. @ 35°C 

Oven temp.: 35°C (hold 2 min.) to 190°C @ 4°C/min. (hold 15 min .) 

(conditions as per ASTM D6159-97) 

Det.: FID @ 200°C 

Instrument: Agilent 5890 

GC_PC01109 

Figure 2 Analyzing ethylene on an alumina column alone results in 

coelutions that prevent quantification of several impurities. 

• 4 • 

Column: Rt ® -Alumina BOND (KCl deactivation), Peak List 

50m, 0.53mm ID, 10.0µm (cat.# 19760) 1. methane 

Sample: 

Inj.: 

ethylene and C1-C5 hydrocarbons 

1µL split, 60mL/min. split vent flow rate 


2. ethane 

3. ethylene 

4. propane 

Inj. temp.: 200°C 

Carrier gas: helium, constant pressure (5.0psi, 34.5kPa) 

Linear velocity: 25.0cm/sec. @ 35°C 

Oven temp.: 35°C (hold 2 min.) to 190°C @ 4°C/min. 

(hold 15 min.) (conditions as per ASTM 

D6159-97) 

Det.: FID @ 200°C 

Instrument: Agilent 5890 

GC_PC01138 

5. cyclopropane 

6. propylene 

7. acetylene 

8. isobutane 

9. propadiene 

10 n-butane 

11. trans-2-butene 

12. 1-butene 

13. isobutylene 

14. cis-2-butene 

15. isopentane 

16. methyl acetylene 

17. n-pentane 

18. 1,3-butadiene 

www.restek.com

Figure 3 All impurities are fully resolved and easily quantifiable when 

using an Rt ® -Alumina BOND/KCl column coupled to an Rtx ® -1 column. 

Column: Rt ® -Alumina BOND/KCl, 50m, 0.53mm ID, Peak List 

10.0µm (cat.# 19760) in series with 1. methane 

Rtx ® -1, 30m, 0.53mm ID, 5.0µm (cat.# 2. ethane 

10179), connected using a Universal 3. ethylene 

Press-Tight ® Connector (cat.# 20401) 4. propane 

Sample: 

Inj.: 

Inj. temp.: 

Carrier gas: 

Linear velocity: 

Oven temp.: 

Det.: 

Instrument: 

Product Listing 

ethylene and C1-C5 hydrocarbons 

1µL split, 60mL/min. split vent flow rate 


200°C 

helium, constant pressure (8.0psi, 

55.2kPa) 25.4cm/sec. @ 35°C 

35°C (hold 2 min.) to 190°C @ 4°C/min. 

(hold 15 min.) (conditions as per ASTM 

D6159-97) 

FID @ 200°C 

Agilent 5890 

GC_PC01110 

Rt ® -Alumina BOND/KCl Columns (see below) 

Rtx ® -1 Columns (fused silica) 

(Crossbond ® 100% dimethyl polysiloxane) 

ID df (µm) temp. limits length cat. # 

0.53mm 5.00 -60 to 270/290°C 30-Meter 10179 

5. cyclopropane 

6. propylene 

7. acetylene 

8. isobutane 

9. propadiene 

10 n-butane 

11. trans-2-butene 

12. 1-butene 

13. isobutylene 

14. cis-2-butene 

15. isopentane 

16. methyl acetylene 

17. n-pentane 

18. 1,3-butadiene 

same retention time, propadiene and n-butane (peaks 9 

and 10) are only partially resolved, and there is a com- 

plete coelution between methyl acetylene and 

n-pentane (peaks 16 and 17). 

By combining an Rt ® -Alumina BOND/KCl column 

with an Rtx ® -1 column, complete resolution for all of 

the compounds of interest can be achieved. The Rtx ® -1 

column supplements the separation achieved on the 

Rt ® -Alumina BOND/KCl column by contributing 

additional selective retention of less polar compounds 

like isobutane, n-butane, and n-pentane. The extra 

retention of these compounds allows for the complete 

separation of the slightly more polar compounds like 

acetylene, propadiene, and methyl acetylene. Figure 3 

shows the analysis of the same ethylene sample spiked 

with hydrocarbons.All of the compounds that are iden- 

tified in the method can now be resolved for accurate 

identification and quantitation. 

When testing for impurities in ethylene using ASTM 

D6159-97, the combination of an Rt ® -Alumina 

BOND/KCl column coupled to an Rtx ® -1 column pro- 

vides the best resolution of the most common hydro- 

carbon contaminants. Ethylene analysis can also be per- 

formed with a single Rt ® -Alumina Bond column, using 

alternate temperature programs. Restek PLOT columns 

are manufactured using a new technology that signifi- 

cantly reduces particle release, extending column life- 

time and giving highly reproducible retention times. 

Restek columns provide reliable results that can be used 

to protect expensive catalysts, make faster process 

adjustments, and improve product yield. 

Next Generation PLOT Columns 

Minimize Particle Release—Safe for Deans Switching Applications 

Restek has developed new procedures to manufacture PLOT columns with 

concentric stabilized adsorption layers. The new generation PLOT columns 

show a constant flow behavior and have significantly improved mechanical 

stability, resulting in easier operation, more reproducible retention times, and 

reduced particle release. 

Rt ® -Alumina BOND/KCl Columns other phases 

(fused silica PLOT) (KCl deactivation) available 


0.32mm 5 to 200°C 30-Meter 19761 

0.32mm 5 to 200°C 50-Meter 19762 

0.53mm 10 to 200°C 30-Meter 19759 

0.53mm 10 to 200°C 50-Meter 19760 

www.restek.com/petro 


• Rt ® -Q-BOND 

• Rt ®-QS-BOND 

• Rt ®-S-BOND 

• Rt ®-U-BOND 

• Rt ®-Alumina BOND/Na2SO4 

• Rt ® -Msieve 5A 

• MXT ® -Msieve 5A 

NEW! 

advanced 

technology 

• 5 • www.restek.com

Foods, Flavors & Fragrances 

Meet New Requirements for Melamine Analysis 

at 1µg/g in Infant Formula 

Using highly reproducible Rxi-5Sil MS columns for GC/MS 

• FDA GC/MS method successfully transferred to 

meet new lower MRL requirements. 

• Highly reproducible retention times on Rxi ® - 

5Sil MS column are key to accurate compound 

identification. 

• Leak-proof integrated guard column reduces 

matrix contamination and extends analytical 

column lifetime. 

Melamine is a nitrogen-rich industrial compound 

used in the manufacture of plastics, flame-resistant 

products, and cleaning agents. It is not a legal food 

additive; however, it has been added to food products 

in order to falsely represent the amount of protein 

present, as protein level is often determined using sim- 

ple, nonspecific nitrogen content assays (Figure 1). 

Melamine is not considered toxic alone at low doses; 

however, illnesses and deaths have been traced to 

exposure to melamine in the presence of cyanuric acid. 

Figure 1 Melamine and related compounds are rich in nitrogen and 

have been used to misrepresent protein levels in some food products. 

Melamine Cyanuric Acid Ammelide Ammeline 

Table I MS conditions (SIM mode). 

Compound tR (min.) Quant. ion* Qual. ion Qual. ion Qual. ion 

cyanuric acid 10.23 345 (100) 330 (36) 346 (30) 347 (15) 

ammelide 11.07 344 (100) 329 (58) 345 (30) 330 (16) 

ammeline 11.76 328 (100) 343 (79) 329 (29) 344 (24) 

melamine 12.31 327 (100) 342 (53) 328 (30) 343 (17) 

benzoguanamine 14.54 316 (100) 331 (68) 332 (20) 330 (9) 

* Expected relative ion ratios from the FDA method. 

In response the escalating health concerns surrounding melamine, the US Food and Drug Administration (FDA) recently set the safety threshold for 

melamine and related compounds in infant foods at 1µg/g. This level is significantly lower than the previously published minimum reporting 

levels (MRLs) for other commodities (10ìg/g in pet foods and 2.5ìg/g in human foods) and has led to an immediate need for more sensitive 

methodology. Here we adapted the FDA GC/MS procedure originally for higher MRL commodities into a highly reproducible method for the 

low level detection required for analyzing infant formula.(1) 

Detailed Procedure for Infant Formula Now Available 

Matrix spikes were prepared in control infant formula at 0.5µg/g, 1µg/g, and 5µg/g (dry formula was prepared according to label instruc- 

tions prior to fortification). Standards were prepared in extracted matrix at on-column concentrations equivalent to those of fortified 

samples, in order to minimize possible matrix effects.(2,3,4) Samples and standards were derivatized according to the FDA method; 

complete preparation details are available at www.restek.com/melamine. 

Analyses were performed on a Shimadzu QP 2010 Plus GC/MS equipped with an AOC 20i+s auto injector and sampler. GC con- 

ditions are shown in the figures; masses analyzed are shown in Table I. An Rxi ® -5Sil MS analytical column with a 5m Integra- 

Guard integrated guard column was used for analysis. The integrated guard column was chosen since it protects the ana- 

lytical column from matrix contamination with no risk of leaking. 

Highly Reproducible Retention Times Assure Accurate Peak IDs 

This method successfully detected melamine and cyanuric acid to the low levels required for the analysis of infant for- 

mula (Figure 2). Highly reproducible chromatographic separation was achieved and was critical for compound 

identification, since several quantitation ions were also found in other peaks. The FDA method requires retention 

times to be within 0.05 minutes for compound identification. This was easily achieved using the Rxi ® -5Sil MS column, 

which produced highly reproducible results even after the approximately 150 injections made during method 

establishment (Table II). 

For the more information 

on melamine analysis visit 

www.restek.com/melamine 

Table II Retention time is critical to accurate peak identification. Highly 

reproducible results were achieved using an Rxi ® -5Sil MS column (n=3). 

Compound Retention time (min.) 

0.5µg/g 1µg/g 5µg/g 

cyanuric acid 10.26 ± 0.05 10.23 ± 0.0006 10.23 ± 0 .001 

ammelide 11.08 ± 0.003 11.07 ± 0.002 11.08 ± 0.003 

ammeline 11.76 ± 0.001 11.76 ± 0.003 11.76 ± 0.002 

melamine 12.31 ± 0.002 12.31 ± 0.000 12.31 ± 0.004 

benzoguanamine 14.54 ± 0.002 14.54 ± 0.001 14.54 ± 0.002 


Figure 2 Analysis of melamine and related compounds in infant 

formula (1µg/g MRL spike level). 

A. Solvent-only standard Melamine and Related 

(0.01µg/mL injection concentration) Analogs as TMS 

Ret. time (min.) Derivatives 

1. cyanuric acid 10.238 

2. ammelide 11.090 

3. ammeline 11.770 

4. melamine 12.318 

5. benzoguanamine 14.553 

1 

2 

3 

4 

8 9 10 11 12 13 14 15 16 17 

GC_FF01094_(A-E) 

B. Matrix spike Melamine and Related 

(0.01µg/mL injection concentration) Analogs as TMS 

Ret. time (min.) Derivatives in Infant 

1. cyanuric acid 10.258 Formula 

2. ammelide 11.073 

3. ammeline 11.760 

4. melamine 12.328 

5. benzoguanamine 14.538 

1 

2 

3 

4 

8 9 10 11 12 13 14 15 16 17 

GC_FF01095_(A-E) 

Column: Rxi ® -5Sil MS, 30m, 0.25mm ID, 0.25µm, w/ 5m Integra-Guard (cat.# 13623-124) 

Instrument: Shimadzu QP 2010 Plus 

Sample: A. Melamine and Related Analogs Stock Solution (cat.# 33253), 

Benzoguanamine (cat.# 33251) as tri-TMS derivatives, 

injection concentration: 0.01µg/mL 

B. infant formula fortified at 1µg/g with Melamine, Related Analogs 

Stock Solution (cat.# 33253), 

Benzoguanamine (cat.# 33251), analyzed as tri-TMS derivatives, 

injection concentration: 0.01µg/mL 

Inj.: 1.0µL splitless (hold 1 min.), 3.5mm splitless inlet liner w/wool (cat.# 22286-200.1) 


Carrier gas: helium, constant flow 

Flow rate: 1mL/min. 

Oven temp.: 75°C to 320°C @ 15°C/min. (hold 4 min.) 

Det: MS 

Transfer line temp.: 290°C 

Ionization: EI 

Mode: SIM (all method ions in table, only quantification ions were plotted) 

3-in-1Technology 

Highest Inertness • Lowest Bleed • Exceptional Reproducibility 

Global RESTEK Advantage • 7 • 

5 

5 

18 min. 

The analysis of melamine and related compounds in 

infant formula is challenging since it has the lowest 

MRL of all commodities and because its high sugar 

content results in significant matrix interferences. Due 

to these factors, reliable retention time identification 

was critical for compound identification. Using the 

Rxi®-5Sil MS column, highly reproducible retention 

times were achieved and target analytes were reliably 

detected at 1µg/g in infant formula. Successful estab- 

lishment of this method for lower level MRL com- 

modities and packaging of all the required compo- 

nents into a single kit with detailed instructions pro- 

vides analytical laboratories with a simple solution for 

meeting new FDA food safety guidelines for 

melamine. 

For complete analytical details, download FFAN1137 

from www.restek.com. 

References 

1. US Food and Drug Administration, October 2008, GC-MS Screen for the 

Presence of Melamine, Ammeline, Ammelide, and Cyanuric Acid, Laboratory 

Information Bulletin No. 4423, http://www.cfsan.fda.gov/~frf/lib4423.html. 

2. C.F. Poole, J. Chromatogr. A 1158, 241-250 (2007). 

3. T. Èajka, K. Maštovská, S.J. Lehotay and J. Hajšlová, 

J. Sep. Sci. 28, 1048-1060 (2005). 

4. K. Maštovská, S.J. Lehotay and M. Anastassiades, 

Anal. Chem. 77, 8129-8137 (2005). 


Save 

Melamine Analysis Kit 10% 

cat. # 33254 (kit) 

Kit includes: 

when you 

buy as a kit 

Column: 

13623-124: Rxi-5Sil MS with 5 meter Integra Guard Column 

Standards: 

33247: 1mL Melamine Stock Standard (1,000µg/mL) 

33248: 1mL Cyanuric Acid Stock Standard (1,000µg/mL) 

33249: 1mL Ammelide Stock Standard (1,000µg/mL) 

33250: 1mL Ammeline Stock Standard (1,000µg/mL) 

33251: 1mL Benzoguanamine Internal Standard (1,000µg/mL) 

33253: 1mL Melamine Mix Standard (1,000µg/mL) 

18 min. Derivatization Reagent: 

35607: BSTFA w/1% TMCS, 25g vial 

Accessories: 

50mL centrifuge tubes, 5-pk. 

13mm, 0.45µm nylon syringe filters, 5-pk. 

Easy-to-follow instructions with procedural check lists to assist 

with laboratory documentation. 

Rxi ® -5Sil MS Columns (fused silica) 

(Crossbond ® , selectivity close to 5% diphenyl/95% dimethyl polysiloxane) 


0.25mm 0.25 -60 to 330/350°C 30-Meter 13623 

5-Meter Fused Silica Guard Columns 

Nominal ID Nominal OD ea. 6-pk. 

0.25mm 0.37 ± 0.04mm 10043 10043-600 

Universal Press-Tight ® Connectors 

Description 5-pk. 25-pk. 100-pk. 

Universal Press-Tight 

Connectors 20400 20401 20402 

phases available 

• Rxi ® -1ms • Rxi ® -35Sil MS 

• Rxi ® -5ms • Rxi ® -17 

• Rxi ® -5Sil MS • Rxi ® -XLB 

• Rxi ® guard/retention gap columns 

www.restek.com

Environmental 

3-Fold Faster Polybrominated Diphenyl Ether (PBDE) 

Using Your Exisiting Instrumentation 

Use new 15m Rtx ® -1614 columns for fast, reliable PBDE analysis. 

• Increase sample throughput with 3-fold faster 

analysis times. 

• Proprietary deactivation reduces thermal break- 

down, giving high BDE-209 response and excel- 

lent peak symmetry for all PBDE congeners. 

• Meets all method criteria and reliably separates 

BDE-49 and BDE-71. 

Polybrominated diphenyl ethers (PBDEs) are highly 

effective flame retardants and have been instrumental 

in protecting both lives and property. Despite these 

benefits, many PBDEs have been banned or are being 

phased out because of concern surrounding negative 

health effects related to the ubiquitous presence of 

PBDEs in humans and the environment. While expo- 

sure mechanisms and pathways are not completely 

understood, bioaccumulation of these lipophilic com- 

pounds is a concern as PBDEs have been linked to toxic, 

neurological, and reproductive problems. Due to a 

growing need to monitor PBDEs in the environment, 

rapid and accurate methods are increasingly in 

demand. 

EPA Method 1614 is commonly used to analyze PBDEs 

in water, soil, sediment, and tissues. This method presents a considerable challenge to 

the analytical column due to the large number of compounds, resolution and peak 

symmetry requirements, and the active nature of BDE-209. The original draft 

method specified a 30 meter column; however, due to a better understanding of the 

challenges presented by the method compounds and performance metrics, shorter 

columns are now acceptable. Short column methodology presents an important 

opportunity for increasing sample throughput, but requires an analytical column 

with high efficiency to separate critical pairs and sufficient sensitivity for active target 

compounds. 

Restek has developed a new 15m Rtx ® -1614 column specifically for the analysis of 

PBDEs and optimized the GC conditions to obtain fast analysis times. As shown in 

Figure 1, compounds are separated in under 20 minutes—3 times faster than typical 

analyses on traditional 30m columns. All method criteria were easily met, including 

the separation of BDE-49 and BDE-71. Resolution of these compounds was 8% 

based on valley height relative to the height of the shortest peak, which is well with- 

in the EPA 1614 requirement of less than 40%. 

In developing the Rtx ® -1614 column, the deactivation was optimized to give a high 

response for BDE-209, which is the most challenging compound in the method. 

BDE-209 readily breaks down due to heat and active sites in both the injection port 

and column. On-column breakdown of BDE-209 is often observed as peak fronting, 

but the deactivation used to manufacture the Rtx ® -1614 column virtually eliminates 

this problem. The high inertness of this column also results in outstanding peak sym- 

metry for all PBDEs, including BDE-99 (as determined by EPA method 1614 tailing 

Figure 1 New 15m Rtx ® -1614 columns meet all method requirements 3 times faster than long column options, sig- 

nificantly improving sample throughput. 

Resolution = 

8.2% 


Excellent resolution 

of BDE-49 (17) & BDE71 (18) 

GC_EV01025 

• 8 • 

No column 

decomposition 

of BDE209 

Symmetry = 

1.2 

See page 9 for peak 

identifications and 

analytical conditions. 

www.restek.com

factor). The high response and excellent symmetry for BDE-209 and other PBDEs 

observed on the Rtx ® -1614 column improves overall accuracy in sample reporting, 

and shortened analysis time allows more samples to be run per hour. 

In conclusion, the new 15m Rtx ® -1614 column is an excellent column for analyzing 

polybrominated diphenyl ethers. Under the conditions shown, all chromatographic 

method criteria can be met in just 20 minutes, compared to 60 minute run times for 

typical longer column methods. Speeding up analysis times by a factor of 3 using the 

15m Rtx ® -1614 column allows labs to significantly increase sample throughput. 

Peak identifications and analytical conditions for Figure 1 (page 8). 

1. BDE-10 15. BDE-37 29. BDE-126 

2. BDE-7 16. BDE-75 30. BDE-154 

3. BDE-8 17. BDE-49 31. BDE-153 

4. BDE-11 18. BDE-71 32. BDE-138 

5. BDE-12 19. BDE-47 33. BDE-166 

6. BDE-13 20. BDE-66 34. BDE-183 

7. BDE-15 21. BDE-77 35. BDE-181 

8. BDE-30 22. BDE-100 36. BDE-190 

9. BDE-32 23. BDE-119 37. BDE-208 

10. BDE-17 24. BDE-99 38. BDE-207 

11. BDE-25 25. BDE-116 39. BDE-206 

12. BDE-28 26. BDE-118 40. BDE-209 

13. BDE-33 27. BDE-85 

14. BDE-35 28. BDE-155 

Column: Rtx ® -1614, 15m, 0.25mm ID, 0.10µm (cat.# 10295) 

Sample: 100-300ppb PBDE PAR Solution (cat.# EO-5113, 

Cambridge Isotope Laboratories Inc.), 

500ppb decabromodiphenyl ether 

(cat.# BDE-209, Wellington Laboratories) 

Inj.: 1µL splitless (hold 1 min.), 4mm cyclo double 

gooseneck liner (cat.# 20896) 


Carrier gas: helium, constant flow 

Linear velocity: 60cm/sec. @ 120°C 

Oven temp.: 120°C (hold 1 min.) to 275°C @ 15°C/min. to 300°C 

@ 5°C/min. (hold 5 min.) 

Detector temp.: µ-ECD @ 345°C 

Restek Electronic 

Leak Detector... 

Go to www.restek.com/leakdetector for details. 


Rtx ® -1614 Columns (fused silica) 

(5% phenyl methyl) 


0.25mm 0.10 -60 to 330/360°C 15-Meter 10296 

0.25mm 0.10 -60 to 330/360°C 30-Meter 10295 

Splitless Liners for Agilent GCs 

ID* x OD & Length 

Cyclo Double Gooseneck (4mm) 

qty. cat.# 

4.0mm x 6.5mm x 78.5mm 


ea. 20895 

4.0mm x 6.5mm x 78.5mm 


5-pk. 20896 

4.0mm x 6.5mm x 78.5mm 25-pk. 20997 

...and Introducing the NEW Restek 

ProFLOW 6000 

Electronic Flowmeter 

NEW! 

Go to www.restek.com/flowmeter for details.

Clinical/Forensic/Toxicology 

5 Minute Analysis of Vitamin D 

in Serum by LC/MS/MS 

Ultra Aqueous C18 column is ideal for high-throughput labs 

• High sensitivity improves low level accuracy in 

matrix. 

• 5 minute analysis time speeds up sample 

throughput. 

• Excellent selectivity for vitamin D minimizes 

matrix interference. 

Monitoring of vitamin D levels in patients is important 

for the prevention and control of disease. Vitamin D, 

specifically 25-hydroxy vitamin D, plays a critical role in 

controlling calcium and phosphate levels in the body. If 

these levels are not adequately controlled, bone condi- 

tions such as rickets in children or osteoporosis in 

adults may occur. 25-hydroxy vitamin D is a hydropho- 

bic, fat soluble vitamin that is absorbed like a fat in the 

intestines. It is commonly used to diagnose conditions 

that interfere with fat absorption, such as Crohn's dis- 

ease. Since vitamin D analysis is one of the most com- 

monly run procedures in clinical labs, high throughput, 

high sensitivity analytical methods are desirable. 

Conventional techniques for vitamin D analysis, based 

on immunoassay or LC/UV, often lack adequate sensi- 

tivity, specificity, and speed; thus, interest in LC/MS/MS 

methods is growing. Here we establish conditions for 

routine vitamin D testing by LC/MS/MS which result in 

highly symmetric peaks that elute in just 5 minutes. 

25-hydroxy vitamin D appears in several forms, but 

vitamin D2 and vitamin D3 are the most commonly 

analyzed. These forms are very similar and differ only in 

one methyl group and a double bond (Figure 1). 

Because 25-hydroxy vitamin D is a hydrophobic 

species, this compound is extremely amenable to 

reverse phase liquid chromatography (RPLC). While 

conventional C18 columns are commonly used in 

RPLC, for this analysis we selected an Ultra Aqueous 

C18 column instead. This phase is more retentive than a 

C18, which helps separate the vitamin D species from 

less retained matrix components. LC/MS/MS analysis 

using an Ultra Aqueous C18 column resulted in excel- 

lent peak shape, which contributes to enhanced sensi- 

tivity (Figure 2). To evaluate retention, human serum 

samples were extracted in acetonitrile and analyzed. 

Both vitamin D analytes were well-separated from 

matrix interferences (Figure 3). 

Analyzing vitamin D by LC/MS/MS using an Ultra 

Aqueous C18 column is an ideal method for high- 

throughput clinical labs interested in accurate low-level 

detection and fast analysis times. Excellent peak shape 

and MS sensitivity result in faster, more accurate analy- 

sis of clinical samples. 

Ultra Aqueous C18, 5µm Columns 

3µm Column, 2.1mm cat. # 

50mm 9178352 


Figure 1 

Structures of 

vitamins D2 and D3. 

Vitamin D2 Vitamin D3 

Figure 2 Ultra Aqueous C18 columns provide outstanding peak symme- 

try for vitamin D, improving accuracy at low concentrations. 

Conditions: 

Instrument: Shimadzu Prominence ® UFLCXR 

Mobile phase: A: 0.1% formic acid in water 

B: 0.1% formic acid in methanol 


0.00 50 

2.5 100 

3.5 100 

3.6 50 

5.00 50 

Flow: 700µL/min. 

Temp.: 40°C 

Det.: Applied Biosystems 3200 QTRAP 

LC/MS/MS system 

Ion source: TurboIonSpray ® , APCI+ 

Mode: MRM 

Dwell time: 100msec. 

Declustering 

Compound Q1 Q3 potential (V) 

d6-25-OH D3 (IS) 389.3 211.2 68 

25-OH D3 383.3 211.2 68 

25-OH D2 395.3 229.2 55 

25-OH D3 383.3 229.2 68 

25-OH D2 395.3 269.2 55 

25-OH D2 395.3 119.0 55 

Ret. Time 

(min.) 

3.00 

3.00 

3.04 

3.00 

3.04 

3.04 

Sample: 

Inj.: 20µL 

Conc.: 20ng/mL vitamin 

D standard 

Column: Ultra Aqueous C18 

Cat.#: 9178352 




Figure 3 Excellent results for vitamin D in patient serum can be 

obtained in just 5 minutes. 

Sample: 

Inj.: 20µL 

Conc.: extracted serum sample 

See Figure 2 for conditions and compound list. 

LC_CF0492 

LC_CF0491 



Re-Developing your Current Impurity 

Assay for High Sample Throughput 

Continued from page 3. 

Find the Column that Fits 

So, how fast is fast enough? Admittedly, the example analyses could be scaled further by using an Ultra II Aromax in a 1.9µm UHPLC format. 

This is certainly an option, but the extra gains will be marginal and the cost of instrumentation may be prohibitive. In this example, we worked 

within the framework of our instrumentation and made a significant impact on the speed and cost of analysis. Each lab must carefully evaluate 

their own analytical needs and resources before determining how fast is fast enough. 

Short-term savings of time and money can easily be 

achieved by optimizing outdated methods with novel 

column chemistries where appropriate. Evaluating 

selectivity is the first step in making strategic column 

choices; additional savings can be realized by choosing 

a particle size that best fits lab needs. New Ultra II 

LC columns are fully scalable, available in many phase 

chemistries, and designed to help labs adapt methods 

and speed up analysis times on any LC system. 

Product Listing NEW! 

Ultra II Aromax Columns (USP L11) 

Physical Characteristics: 

particle size: 2.2µm, 3µm or 5µm, spherical 

pore size: 100Å 

carbon load: 17% 

endcap: fully endcapped 

pH range: 2.5 to 7.5 

temperature limit: 80°C 

Chromatographic Properties: 

Ultra II Aromax is a unique reversed phase material that 

exhibits superior retention and selectivity for aromatic 

and/or unsaturated compounds, compared to conventional 

alkyl and phenyl phases. This column is a great alternative 

to our Biphenyl phase when increased retention is required. 

A very suitable choice for analysis of steroids, tetracyclines, 

drug metabolites, and other compounds that contain some 

degree of unsaturation. 

Available Phases: 

• C18 • C8 

• Biphenyl • PFP Propyl 

• Aromax • Aqueous C18 

• Silica 

Table I Savings comparison of famotidine impurity assays based 

on column choice. 

5µm Conv. C18 5µm Ultra II 3µm Ultra II 2.2µm Ultra II 

(USP) Aromax Aromax Aromax 

(250mm x 4.6mm) (150mm x 4.6mm) (100mm x 3.2mm) (50mm x 3.0mm) 

Analysis Time 45 min. 15 min. 8 min. 4 min. 

Equilibration Time 15 min. 4 min. 3 min. 1.5 min. 

Total Run Time 60 min. 19 min. 11 min. 5.5 min. 

% Time Savings — 68% 82% 91% 

Solvent acetonitrile methanol methanol methanol 

Solvent Volume 90mL 38mL 13.2mL 6.6mL 

% Solvent Savings — 67% 82% 91% 

Savings calculated relative to original USP method. 

1.0mm ID 2.1mm ID 3.0mm ID 3.2mm ID 4.6mm ID 

Length cat.# cat.# cat.# cat.# cat.# 

2.2µm Columns 

30mm 9607832 9607833 

50mm 9607852 9607853 

100mm 9607812 9607813 

3µm Columns 

30mm 9607331 9607332 — 9607333 9607335 

50mm 9607351 9607352 — 9607353 9607355 

100mm 9607311 9607312 — 9607313 9607315 

150mm 9607361 9607362 — 9607363 9607365 

5µm Columns 

30mm 9607531 9607532 — 9607533 9607535 

50mm 9607551 9607552 — 9607553 9607555 

100mm 9607511 9607512 — 9607513 9607515 

150mm 9607561 9607562 — 9607563 9607565 

200mm 9607521 9607522 — 9607523 9607525 

250mm 9607571 9607572 — 9607573 9607575 

Guard cartridges are available, visit our website at www.restek.com for ordering information. 

Available Particle Sizes: 

• 1.9µm for UHPLC 

• 2.2µm for UFLC and RRLC 

• 3µm and 5µm for HPLC 

www.restek.com/ultra2 

More phases coming soon! 

Universal application for Any 

LC System, providing scalability 

and unsurpassed selectivity on 

a wide range of particle sizes. 

NEW! 

Global RESTEK Advantage • 11 • www.restek.com

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