LC/MS Compatible Separation of on ZirChrom®-MS

Clayton McNeff, Ph.D., Bingwen Yan, Ph.D. and Steven Rupp ZirChrom Separations, Inc.

Technical Bulletin # 296

The chromatography of basic drugs on C18-silica phases is Flow Rate: 1.0 ml/min. often complicated due to mixed-mode interactions that cause Injection Vol.: 1 µl poor peak shape and irreproducible results. ZirChrom®-MS is Pressure Drop: 59 bar a new zirconia-based reversed-phase column that has enhanced Detection: UV at 254 nm mixed mode retention characteristics which allow for LC/MS chromatography of highly basic amines with excellent peak Five basic pharmaceutical compounds were separated using simple shape and efficiency. acetonitrile/water isocratic elution and a LC/MS friendly acetate buffer. The selectivity and peak symmetry of all five compounds is O

N S very good which allows for a separation using only a short 5 cm N long column.

N

Doxepin Methapyrilene N 1 Br 2 3

N 5 4 N

Absorbance (mAU)

N N H

Figure 1: Structures for the Compounds of Interest. Time (minutes)

Introduction The chromatography of basic drugs on C18-silica phases is difficult Figure 2: Separation of 1=Methapyrilene, 2=, due to interactions between silanols and the amines1. We have 3=Brompheniramine, 4=Amitriptyline, and 5=Nortriptyline. found that a , Amitriptyline, may be used as a probe solute for quantifying silanophilicity of HPLC columns. The surface chemistry of zirconia-based phases is dominated by This method can be tailored to your specific application needs. Lewis acid sites, rather than the Bronsted acid sites, which dominate ZirChrom method developers can help to optimize and transfer this the surface chemistry of silica phases. The mixed-mode retention method to your site. Please contact ZirChrom technical support at character of ZirChrom®-MS (cation-exchange and reversed-phase) 1-866-STABLE-1 or [email protected] for details. allows separations that were previously difficult using conventional silica C18 phases. This application note shows an impressive ZirChrom phases offer unique selectivity, high efficiency, and LC/MS compatible separation of antidepressants in a highly excellent chemical and thermal stability. organic, near neutral pH mobile phase. Experimental References A mixture of five basic drugs (four antidepressants) was separated 1) G.B. Cox, J. Chromatography A. 656, 353, 1993. ® at 35 ºC using a ZirChrom -MS column. The separation conditions were as follows: ZirChrom Separations, Inc. Column: ZirChrom®-MS, 50 mm x 4.6 mm i.d. 617 Pierce Street, Anoka, MN 55303 (Part Number: MS01-0546) 1-866-STABLE-1 Mobile Phase: Isocratic elution: 65/35 A/B [email protected] A: acetonitrile B: 10mM ammonium acetate, pH 5.0 Visit www.zirchrom.com for more application notes using ultra- Temperature: 35 ºC stable, high efficiency ZirChrom columns.

LC/MS Compatible Separation of Non-Steroidal Anti-Inflammatory Drugs

® on ZirChrom -MS

Clayton McNeff, Ph.D., Bingwen Yan, Ph.D. and Steven Rupp ZirChrom Separations, Inc.

Technical Bulletin #297

The Lewis acidity of zirconia-based supports for HPLC Mobile Phase: Isocratic elution: 40/60 A/B has historically presented problems in the analysis of A: acetonitrile analytes containing Lewis base moieties, such as B: 10mM ammonium acetate, pH 5.0 carboxylates, particularly in LC/MS applications where Temperature: 35 ºC volatile mobile phase additives are required. In this Flow Rate: 1.0 ml/min. Injection Vol.: 5 µl application note we demonstrate the utility of a new Pressure Drop: 68 bar Lewis acid deactivated zirconia-based column, Detection: UV at 254 nm  ZirChrom -MS. Four non-steroidal anti-inflammatory drugs were separated using O O simple acetonitrile/water isocratic elution and a LC/MS friendly acetate buffer. The selectivity of all four compounds is excellent HN which allows for a very good separation using only a short 5 cm column.

OH 2

OH O 4 Acetaminophen Ketoprofen 1 3 OH

O Ibuprofen O O

Absorbance (mAU) 5 Naproxen OH

Figure 1: Structures of Non-steroidal anti-inflammatory drugs. Introduction Time (minutes) Historically, the Lewis base carboxylic acid moiety on non-steroidal anti-inflammatory drugs required the use of a Lewis base mobile Figure 2: Separation of 1=Acetaminophen, 2=Ketoprofen, phase additive of a higher strength in the elutropic series (such 3=Naproxen, 4=Ibuprofen, and 5=Impurity. as phosphate or fluoride) (1). While these types of additives work well in applications with UV/Vis detection, their use is This method can be tailored to your specific application needs. almost entirely prohibited in LC/MS applications due to their ZirChrom method developers can help to optimize and transfer this relatively low volatility. method to your site. Please contact ZirChrom technical support at 1-866-STABLE-1 or [email protected] for details. The deactivation of Lewis acid sites on the surface of the ® ZirChrom -MS particle allows the chromatography of Lewis References base analytes using mobile phase additives of the users choice including conventional LC/MS compatible buffers (such as (1) Blackwell, J. A.; Carr, P. W., Journal of Liquid acetate and formate) throughout the pH range of 1-10. Chromatography, 14, 2875-2889, 1991.

Experimental Four non-steroidal anti-inflammatory drugs were separated at 35ºC ZirChrom Separations, Inc. ® using a ZirChrom -MS column. The separation conditions were as 617 Pierce Street, Anoka, MN 55303 follows: 1-866-STABLE-1 Column: ZirChrom®-MS, 50 mm x 4.6 mm i.d. [email protected] (Part Number: MS01-0546) Visit www.zirchrom.com for more application notes using ultra- stable, high efficiency ZirChrom columns.

LC/MS Compatible Separation of Beta-Blockers on ZirChrom®-MS

Clayton McNeff, Ph.D., Bingwen Yan, Ph.D. and Steven Rupp ZirChrom Separations, Inc.

Technical Bulletin # 298

The chromatography of basic pharmaceuticals (with amine Flow Rate: 1.0 ml/min. functionalities) on C18-silica phases can be difficult due to Injection Vol.: 5 µl secondary interactions that cause tailed peaks in the neutral pH Pressure Drop: 59 bar range where most silica phases are stable1. ZirChrom®-MS is a Detection: UV at 254 nm new zirconia-based reversed-phase column that has mixed mode retention characteristics which allow for LC/MS Five Beta-Blockers were separated using simple acetonitrile/water chromatography of highly basic amines with excellent peak isocratic elution and an LC/MS friendly acetate buffer. The shape and efficiency. selectivity of all five compounds is excellent using only a short 5 cm long column.

NH2

O NH N N O 2 H Atenolol OH 5 O Lidocaine O 3 1 4

N O H Metoprolol OH Absorbance (mAU) O

O NH ON H Time (minutes) Oxprenolol Alprenolol OH

Figure 2: Separation of 1= Lidocaine, 2= Atenolol, 3= Metoprolol, Figure 1: Structures for the Compounds of Interest. 4= Oxprenolol, and 5= Alprenolol. Introduction This method can be tailored to your specific application needs. The chromatography of basic drugs on C18-silica phases has ZirChrom method developers can help to optimize and transfer this traditionally been problematic. The surface chemistry of zirconia- method to your site. Please contact ZirChrom technical support at based phases is dominated by Lewis acid sites, rather than the 1-866-STABLE-1 or [email protected] for details. Bronsted acid sites, which dominate the surface chemistry of silica phases. The mixed-mode retention character of ZirChrom®-MS ZirChrom phases offer unique selectivity, high efficiency, and (cation-exchange and reversed-phase) allows separations that were excellent chemical and thermal stability. previously difficult using conventional silica C18 phases. This application note shows an impressive LC/MS compatible separation of beta-blockers in a highly organic, near neutral pH mobile phase. References Experimental 1) G.B. Cox, J. Chromatography A. 656, 353, 1993. A mixture of five amine-containing compounds was separated using a ZirChrom®-MS column at 35 ºC. The separation conditions were as follows: ZirChrom Separations, Inc. Column: ZirChrom®-MS, 50 mm x 4.6 mm i.d. 617 Pierce Street, Anoka, MN 55303 (Part Number: MS01-0546) Mobile Phase: Isocratic elution: 65/35 A/B 1-866-STABLE-1 A: acetonitrile [email protected] B: 10mM ammonium acetate, pH 5.0 Temperature: 35 ºC Visit www.zirchrom.com for more application notes using ultra-stable, high efficiency ZirChrom columns.

ZirChrom®-MS Exhibits Unique Selectivity for Basic Pharmaceuticals

Clayton McNeff, Ph.D., Bingwen Yan, Ph.D. and Steven Rupp ZirChrom Separations, Inc.

Technical Bulletin #299

Basic pharmaceuticals are well-known problematic compounds 4.00 on silica C18 due to the interactions between the amine R2 = 0.340 functionalities and non-bonded residual silanol groups1. In this 3.50 application note we demonstrate the utility of a new Lewis acid 3.00 deactivated zirconia-based column, ZirChrom-MS. The new ZirChrom-MS column exhibits unique selectivity for the 2.50 specific set of amine-containing compounds studied. 2.00

Introduction ZirChrom-MS ln k' 1.50 1.00 The chromatography of basic pharmaceuticals on silica C18 has traditionally been so problematic that amitriptyline is commonly -1.50 -1.00 -0.50 0.00 0.50 1.00 used as a probe solute for quantifying silanophilicity of silica ln k' Silica C18 phases. The surface chemistry of zirconia-based phases is dominated by Lewis acid sites, rather than the Bronsted acid sites, Figure 1: Selectivity Comparison for a Set of Basic which dominate the surface chemistry of silica phases. The mixed- Pharmaceuticals - leading Silica C18 versus ZirChrom®-MS. mode retention character of ZirChrom®-MS (cation-exchange and reversed-phase) allows separations that were previously difficult As a result of the mixed-mode ion-exchange and reversed-phase using conventional silica C18 phases. characteristics of ZirChrom®-MS, the elution order of basic pharmaceuticals is often quite different compared to leading Using traditional, near normal pH operating conditions one reversed-phase silica phases. Figure 1 shows a plot of ln k’ for typically obtains significantly higher retention for basic compounds eight common basic pharmaceuticals on a leading silica C18 phase ® on ZirChrom -MS versus traditional silica C18. Even though there versus ln k’ for the same compounds on ZirChrom®-MS. There is is much higher carbon loading to silica-based columns the strong no apparent correlation of the retention for these compounds on the ion-exchange contribution to retention results in an overall higher silica C18 phase with the retention on ZirChrom®-MS. This ® retention factor on the ZirChrom -MS column. In fact, the different selectivity is particularly useful in method development for ® ZirChrom -MS phase has relatively higher retention for basic drugs basic pharmaceuticals. When a pair of basic compounds cannot be compared to all of the zirconia-based reversed phases as well. In separated using a traditional silica C18 phase, the chances of them general, excellent peak shapes may be obtained using LC/MS separating on ZirChrom®-MS are much better than on any other compatible, near neutral pH operating conditions. silica phase.

In addition, ZirChrom®-MS enables the user to analyze basic This method can be tailored to your specific application needs. pharmaceutical compounds, acidic pharmaceutical compounds, or ZirChrom method developers can help to optimize and transfer this both simultaneously, under LC/MS compatible, near neutral pH method to your site. Please contact ZirChrom technical support at operating conditions. 1-866-STABLE-1 or [email protected] for details.

Experimental ZirChrom phases offer unique selectivity, high efficiency, and excellent chemical and thermal stability. The selectivity of a set of basic pharmaceuticals was compared ® using a leading silica C18 column and a ZirChrom -MS column. References The separation conditions were as follows: 1) G.B. Cox, J. Chromatography A. 656, 353, 1993. Column Size: 50 mm x 4.6 mm i.d.

Mobile Phase: Isocratic elution: 65/35 A/B

A: methanol B: 25mM ammonium phosphate, pH 6.0 ZirChrom Separations, Inc. Temperature: 35 ºC 617 Pierce Street, Anoka, MN 55303 Flow Rate: 1.0 ml/min. 1-866-STABLE-1 Injection Vol.: 5 µl [email protected] Detection: UV at 254 nm Solutes: (From left to right in Figure 1) Visit www.zirchrom.com for more application notes using ultra- Methapyrilene, Pyrilamine, , stable, high efficiency ZirChrom columns. Brompheniramine, , Nortryptyline, Doxepin, Amitryptyline

The Analysis of Basic Compounds Using Neutral pH Conditions:

A Column Comparison Study

Clayton McNeff, Ph.D., Bingwen Yan, Ph.D., and Steven Rupp ZirChrom Separations, Inc.

Technical Bulletin # 303

ZirChrom®-MS represents another first of its kind zirconia- The following 26 compounds were included in the test set: column designed specifically for MS detection and the high (1) Methapyrilene, (2) Pyrilamine, (3) Tripeleneamine, demands of pharmaceutical method development specifications. (4) Chlorpheniramine, (5) Brompheniramine, (6) Thiothixene, Using our novel covalently attached Lewis acid deactivation (7) Doxepin, (8) Amitryptyline, (9) Desipramine, chemistry, ZirChrom has developed a highly retentive reversed- (10) Nortryptyline, (11) , (12) , (13) Lidocaine, phase HPLC column, which is easy to use and which still has (14) Atenolol, (15) Metoprolol, (16) Oxprenolol, (17) Alprenolol, the inherent chemical stability advantages of zirconia-based (18) , (19) 4-chlorophenol, (20) Acetaminophen, HPLC columns. Most importantly this new column still (21) Ketoprofen, (22) Ibuprofen, (23) Naproxen, (24) Toluene, maintains the very different chromatographic selectivity, (25) Biphenyl, (26) Phenanthrene. especially for basic pharmaceuticals that zirconia-based columns are well known to have compared to traditional Compounds (1) thru (17) are basic; compounds (18) thru (23) are bonded C18 silica phases. This new column compliments the acidic; and compounds (24) thru (26) are neutral test probes. family of reversed phase columns that ZirChrom currently ® markets; a family of chemically different and thermally stable Retention Comparison of ZirChrom -MS Versus HPLC columns. a Leading Bonded Phase C18 Silica Introduction 20 for Basic Compounds ZirChrom®-MS is a surface deactivated, reversed-phase zirconia 18 column designed specifically for LC-MS applications, particularly 16 14 those involving basic pharmaceutical compounds. The following 12 unique features make ZirChrom®-MS an ideal choice for today’s 10 LC-MS method developer: k' 8 1. Compatible with volatile, near neutral pH mobile phase buffers 6 including ammonium acetate and formate. 4 2 2. Enhanced retention for basic pharmaceutical compounds 0 compared to bonded phase C18 silica under LC-MS 1113231467514151681217910 compatible operating conditions. solute number 3. Very different chromatographic selectivity for basic drugs Figure 1. Retention Comparison for Basic Compounds. compared to bonded phase C18 silica using LC-MS conditions. ® 4. Improved peak shape and efficiency for basic drugs compared Figure 1 shows a retention comparison of ZirChrom -MS versus a to bonded phase C18 silica using LC-MS conditions. leading bonded phase C18 silica for basic compounds under these LC-MS compatible operating conditions. For illustrative purposes 5. The ability to analyze basic, acidic or neutral pharmaceutical the solutes are organized in order of increasing retention on compounds, or mixtures of all three, simultaneously. ZirChrom®-MS. This figure demonstrates that ZirChrom®-MS 6. Low column bleed characteristics due to covalent bonding offer enhanced retention for basic pharmaceutical compounds chemistry. compared to bonded phase C18 silica. ® Experimental Selectivity Comparison of ZirChrom -MS Versus A column comparison study using pharmaceutically relevant a Leading Bonded Phase C18 Silica compounds was performed to demonstrate the unique for Basic Compounds ® characteristics and excellent performance of ZirChrom -MS 20.0 relative to a leading bonded phase C18 silica column. The 18.0 separation conditions were as follows: 16.0 R2 = 0.067

-MS 14.0

® Columns: ZirChrom®-MS (Part Number: MS01-0546) 12.0 50 mm x 4.6 mm i.d., 3µm particle size; 10.0 8.0 Leading bonded phase C18 silica, 6.0 150 mm x 4.6 mm i.d., 3.5 µm particle size

k' ZirChrom 4.0 Mobile Phase: Machine-mixed 80/20 ACN/10 mM ammonium 2.0 acetate, pH=6.7 without pH adjustment 0.0 Temperature: 35 ºC 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Flow Rate: 1.0 ml/min. k' C18 Injection: 0.1 µl Detection: UV at 254 nm Figure 2. Selectivity Comparison for Basic Compounds. ® Figure 2 shows a selectivity comparison of ZirChrom -MS versus a mAU leading bonded phase C18 silica for basic compounds under these A ® LC-MS compatible operating conditions. This figure demonstrates B ZirChrom -MS that ZirChrom®-MS offers very different chromatographic C selectivity (R2 = 0.067) for basic drugs compared to bonded phase C18 silica. D E Efficiency Comparison of ZirChrom®-MS Versus a Leading Bonded Phase C18 Silica

200,000 Basic

150,000 Acidic Neutral

min. 100,000 Figure 5. ZirChrom®-MS Separation of Basic Compounds. N / meter 50,000 Elution Order: (A) Methapyrilene, (B) Brompheniramine, (C) Doxepin, (D) Amitriptyline, (E) Nortryptyline. 0 Note: Column used was 150 mm x 4.6 mm i.d., 3µm particle size. 1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526

solute number mAU C Figure 3. Efficiency Comparison for All Compounds. C18 Silica Figure 3 shows the efficiency comparison of ZirChrom®-MS versus a leading bonded phase C18 silica for all compounds under these E LC-MS compatible operating conditions. ZirChrom®-MS produced superior column efficiency (plates per meter) in 16 out of 17 cases involving basic compounds. The leading bonded phase C18 silica A D only produced acceptable column efficiency in the cases involving acidic and neutral compounds. B

Symmetry Comparison of ZirChrom®-MS Versus a Leading Bonded Phase C18 Silica min.

C18 Silica Exhibits 4.00 Figure 6. Leading Bonded Phase C18 Silica Separation. Severe Tailing Basic 3.50 Note: The basic compounds are lettered the same as in Figure 5. 3.00 Acidic 2.50 Neutral Figures 5 & 6 show a representative separation involving some of ® 2.00 the basic compounds used in the study. Clearly, ZirChrom -MS 1.50 offers both unique selectivity and superior chromatographic 1.00 performance relative to a leading bonded phase C18 silica for basic 0.50 compounds under these LC-MS compatible operating conditions.

tailing factor [1/symmetry] 0.00 1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526 Summary solute number ® Figure 4. Symmetry Comparison for All Compounds. In summary, ZirChrom -MS consistently outperformed a leading bonded phase C18 silica for the separation of basic compounds under LC-MS compatible operating conditions. ZirChrom®-MS (Note: tailing factor was calculated by the formula [1/symmetry] produced enhanced retention, unique selectivity, greater efficiency using the symmetry value as reported by the Agilent® 1100 and improved symmetry for virtually all of the basic compounds Chemstation® software.) that were studied. Figure 4 shows the symmetry comparison of ZirChrom®-MS versus a leading bonded phase C18 silica for all compounds under these ZirChrom Separations, Inc. LC-MS compatible operating conditions. ZirChrom®-MS produced 617 Pierce Street, Anoka, MN 55303 superior column symmetry in 16 out of 17 cases involving basic 1-866-STABLE-1 compounds. The leading bonded phase C18 silica only produced [email protected] acceptable column symmetry in the cases involving acidic and neutral compounds. Visit www.zirchrom.com for more application notes using ultra-

stable, high efficiency ZirChrom columns. Technical Bulletin #304

G&T SEPTECH MICRO-HPLC PRODUCTS HotSep® ZirChrom-MS® micro-HPLC Columns

Naphtalene Fluorene 8 Toluene 50 x 0.3 mm i.d. 6 N/m: 102,000-122,000 5.54, Fluorene

4 Acetone 3.49, Naphtalene 1.35, Aceton As: 1.01-1.06 2.66, Toluen

Intensity (mV) 2 Part # MS01-0503 (3 µm)

0

0 1 2 3 4 5 6 7

Retention Naphtalene Time (min) 10 Fluorene

Toluene 8 50 x 0.5 mm i.d.

6 N/m: 104,000-113,000 1.02, Aceton Acetone 5.05, Fluorene 3.09, Naphtalene As: 1.06-1.14 4 2.27, Toluen Intensity (mV)

2 Part # MS01-0505 (3 µm)

0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Retention Time (min) Naphtalene 5 Highly efficient, LC/MS compatible Toluene Fluorene micro-HPLC columns with different 4 Acetone 50 x 1.0 mm i.d. inner diameters: 3

2 N/m: 124,000-126,000 (A) 0.3 mm ID, flow rate: 3 µL/min 3.25, Naphtalene 2.33, Toluen (B) 0.5 mm ID, flow rate: 8 µL/min 0.92, Aceton 5.47, Fluorene As: 0.98-1.04 1

Intensity (mV) (C) 1.0 mm ID, flow rate: 30 µL/min 0 Part # MS01-0510 (3 µm) Mobile phase: ACN – water (70:30, -1 v/v). UV-detection at 254 nm.

0 1 2 3 4 5 6 7 Retention Time (min)