constant flow rate. The GC oven was

constant flow rate. The GC oven was initially maintained for 1.5 min
at 70
◦
C, and then the temperature was ramped to 180
◦
C at
80
◦
C/min, then increased to 250
◦
C at 40
◦
C/min, and finally taken
to 290
◦
C at 70
◦
C/min and held for 4.5 min. The MS transfer line
was 250
◦
C, the ion source was set at 320
◦
C, and quadrupole temperatures were 150
◦
C. An Agilent multimode inlet was used for
injection; a temperature program of 80
◦
C held for 0.31 min with
vent open at 50 mL/min He flow, then closed (for 3 min) and ramped
to 420
◦
C at 320
◦
C/min, and held until the end of the 9.7 min analysis. The injection volume was 5 L. Before each sequence, a full
autotune of the MS was conducted, and between injections, the
10 L syringe was washed 8 times with an equal parts mixture of
acetone/MeCN/water/MeOH followed by 8 rinses with MeCN.
2.4. LC-MS/MS conditions
The LC-MS/MS analyses were conducted as in previous studies
[15,18,21] using an Agilent 1100 LC coupled to an Applied Biosystems (Toronto, ON, Canada) API-3000 triple quadrupole MS. Analyst
software version 1.6 was used for instrument control and data
processing. The LC separation was carried out at 30
◦
C on a Prodigy
ODS-3 150 mm × 3 mm i.d., 5 m particle size analytical column
coupled to a C184 mm × 3 mm i.d. guard column, both from Phenomenex. The mobile phase consisted of (A) water and (B) MeCN both
containing 0.1% formic acid, and flow rate was 0.3 mL/min. Gradient
elution was used with an initial starting condition of 30% B which
was increased to 100% over 8 min, and held for 6 min to yield total
run time of 14 min. Injection volume was 10 L. MS/MS analyses
were performed using electrospray ionization in the positive mode
with a dwell time of 20 ms for all pesticides. The source temperature
and ion spray voltage were set at 525
◦
C and 4500 V, respectively,
and the curtain and nebulizer gases were 11 and 14 psi, respectively.
2.5. Sample preparation
The following 4 versions of QuEChERS were evaluated in this
work: (A) AOAC Official Method 2007.01 (buffered); (B) NH4Cl
78 M.Á. González-Curbelo et al. / J. Chromatogr. A 1358 (2014) 75–84
salt-out (unbuffered); (C) NH4O2CH (buffered); and (D) NH4OAc
(buffered). Each method entailed the following steps: (1) 15 g
sample (5 g wheat grain) was placed into a 50 mL polypropylene
centrifuge tube, and fortifications were made to yield 10, 75 and
400 ng/g for the pesticide analytes and 200 ng/g for the IS (the
tubes were vortexed for 1 min to better integrate the spikes into
the samples); (2) 15 mL of MeCN containing 1% (v/v) HOAc were
added for both the AOAC 2007.01 and NH4OAc versions, or 15 mL
of MeCN containing 5% (v/v) formic acid for the NH4O2CH version, or 15 mL of MeCN for the NH4Cl version; (3) the tubes were
shaken vigorously by hand for 1 min (for wheat, 15 mL water was
also added and samples were shaken for 1 h in accordance with
a previous study [22] using an automated shaker); (4) 6 g of anh.
MgSO4plus 1.5 g of anh. NaOAc were added for the AOAC 2007.01
version, or 7.5 g of NH4Cl, NH4O2CH or NH4OAc for the other
respective versions; (5) the tubes were vigorously shaken again
for 1 min by hand and centrifuged at 3700 rcf for 1 min; (6) 1 mL
of the MeCN extract was transferred to a d-SPE tube containing
150 mg anh. MgSO4, 50 mg PSA (150 mg for wheat), 50 mg C18, and
7.5 mg GCB, vortexed for 30 s, and centrifuged at 3700 rcf for
1 min; (7) 0.5 mL of the extracts were transferred to the appropriately labeled autosampler vials, and 50 L (17 L for wheat) of
the QC solution was added (to make all sample volumes equal to
the matrix-matched calibration standards, 50 or 17 L of MeCN
was also added as needed); (8) for LPGC-MS/MS, 200 L of each
extract was transferred into a second autosampler vial containing
deactivated glass inserts as well as 20 L of the analyte protectants solution; (9) for LC-MS/MS, 600 L of 0.1% aqueous formic
acid solution was added to the 300 L of extract remaining in
the first vial. For lemon, a precipitate appeared after dilution with
water, and all of its final extracts were filtered through 0.45 m
PVDF Mini-UniPrep filter vials from Whatman (Florham Park, NJ,
USA).
Matrix-matched calibration involved the same steps as above
using blank extracts, except the d-SPE step (6) was scaled up
to 8 mL in 15 mL tubes and no spike nor IS solutions were
added until step (7), when 50 L of calibration standard solution (17 L for wheat grain) was added to the extracts in the
autosampler vials. Fenthion-d6was used as the IS for LPGC-MS/MS
and atrazine-d5for LC-MS/MS. In the case of the final NH4O2CH
buffered method, additional validation experiments were conducted, and the results were compared using the d-SPE step (6)
or not.
3. Results and discussion
As shown in Table 1, a total of 48 targeted chemicals including
IS, QC, isomers, adducts, and degradation products were monitored in both LPGC-MS/MS and LC-MS/MS with 15 overlapping. The
aldicarb Na-adduct was monitored out of curiosity in the study, and
tetrahydrophthalimide (THPI) and phthalimide (PI) are the degradation products of captan and folpet, respectively. Even though
only the parent analytes were added to the mix, the THPI and
PI could be semi-quantified from their presence in the calibration standards. However, captan and folpet were strictly treated
as the pesticide analytes, not THPI and PI. Also, the spinosad reference standard consists of 85% spinosyn A and 15% spinosyn D,
and although both were monitored and independently reported,
they were treated as a single pesticide analyte (spinosad). The
sulfone metabolites of aldicarb and fenthion were each prepared
from separate standards, and they were all treated as unique
analytes. The 4 isomers of cypermethrin were integrated as a single peak. Thus, there were 43 distinct pesticide analytes in the
method.
The pesticides were carefully selected to cover a wide range
of representative analytes of different chemical classes commonly
monitored in many labs worldwide. With a few minor differences, these have been used in previous studies to demonstrate
method performance [6,15,18], and it has been demonstrated that
when acceptable results for these pesticides are achieved, then the
method yields similar quality of results for hundreds of other pesticides of the same properties [6,7,23]. Indeed, quality assurance
guidelines recommend the use of representative pesticides and
matrices such as these for quality control spikes during routine
analyses [24].
3.1. Optimization of the acid/ammonium salt ratio for buffering
In the previous study by Nanita and Padivitage [11], ammonium
chloride, acetate, formate, carbonate, and sulfate were evaluated
to select the best salting out reagent for extraction in QuEChERS prior to FI-MS/MS. Only 14 pesticides were monitored, all
of which were LC-amenable (none of which were GC-only compatible), and buffering was not conducted. NH4Cl, NH4O2CH, and
NH4OAc all gave similarly acceptable results in the experiments,
and NH4Cl was ultimately selected mainly due to the minor practical consideration of a slightly lower cost. However, NH4Cl actually
has rather high melting and boiling points of 338 and 520
◦
C,
respectively, and sublimes to form ammonia and HCl. On the
contrary, NH4O2CH, and NH4OAc have melting points of only
110–117
◦
C and boiling points of 165–180
◦
C, and upon heating,
they degrade to water plus formamide and acetamide, respectively, rather than direct generation of more corrosive products.
Furthermore, buffering at pH 4–5 in QuEChERS had been previously demonstrated to yield higher recoveries for pH-dependent
pesticides [7,15], and initial experiments were designed to determine the amount of HOAc and formic acid that should be used to
achieve a suitable pH in combination with their respective ammonium salts.
In this experiment, 15 g of apple and lemon samples were separately extracted using 15 mL of MeCN with increasing amounts of
HOAc or formic acid (1, 2, 3, 5, 7.5 or 10%, v/v) plus 7.5 g of NH4OAc
or NH4O2CH, respectively (we used 1:1 sample:solvent ratio and
excess solids rather than saturated aqueous solutions employed by
Nanita and Padivitage [11]). After agitation and centrifugation, the
two phases were separated and pH was measured in both layers
including 4-fold dilution with deionized water for the MeCN phase
as done previously [15]. Fig. 1 shows the pH results from the experiment. As expected, the formate buffer (pKa= 3.75) provided lower
pH than the acetate buffer (pKa= 4.75), and good consistency for
both matrices. The addition of 5% formic acid yielded a pH value
around 3 in the MeCN phase and

Hasil (Bahasa Indonesia) 1:

laju aliran konstan. GC oven awalnya dipertahankan selama 1,5 minpada 70◦C, dan kemudian suhu menggenjot-180◦C pada80◦C/menit, kemudian ditingkatkan menjadi 250◦C pada 40◦C/menit, dan akhirnya diambiluntuk 290◦C pada 70◦C/menit dan berlangsung selama 4,5 min. MS mentransfer barisadalah 250◦C, sumber ion didirikan di 320◦C, dan suhu quadrupole adalah 150◦C. Agilent multimode inlet digunakan untukinjeksi; program temperatur 80◦C diadakan untuk 0.31 min denganlubang terbuka pada 50 mL/menit dia mengalir, kemudian ditutup (untuk 3 menit) dan menggenjotuntuk 420◦C pada 320◦C/menit, dan berlangsung sampai akhir analisis 9.7 min. Volume injeksi adalah 5 L. Sebelum setiap urutan, penuhautotune dari MS dilakukan, dan antara suntikan,Jarum suntik 10 L dicuci 8 kali dengan bagian yang sama campuranaseton/MeCN/air/MeOH diikuti oleh 8 bilasan dengan MeCN.2.4. LC-MS/MS kondisiLC-MS/MS analisis dilakukan dalam studi sebelumnya[15,18,21] menggunakan Agilent 1100 LC digabungkan ke diterapkan Biosystems (Toronto, ON, Kanada) API-3000 triple quadrupole MS. AnalystSoftware Versi 1.6 digunakan untuk data dan kontrol instrumenpengolahan. Pemisahan LC dilaksanakan di 30◦C pada keajaiban150 BPO-3 mm × 3 mm id, 5 m partikel ukuran analitis kolomdigabungkan ke C184 mm × 3 mm id penjaga kolom, baik dari Phenomenex. Fase mobile terdiri dari (A) air dan (B) MeCNberisi 0,1% formic asam, dan laju aliran adalah 0.3 mL/min. gradienelution digunakan dengan kondisi awal awal 30% B yangmeningkat menjadi 100% lebih dari 8 menit, dan berlangsung selama 6 menit untuk menghasilkan totalmenjalankan waktu 14 min. volume injeksi adalah 10 L. MS/MS analisisyang dilakukan menggunakan electrospray ionisasi dalam modus positifdengan waktu tinggal 20 ms untuk semua pestisida. Suhu sumberdan ion semprot tegangan pada 525◦C dan 4500 V, masing-masing,dan tirai dan nebulizer gas yang 11 dan 14 psi, masing-masing.2.5. preparasi sampleVersi berikut QuEChERS 4 sedang dievaluasi dalam hal inibekerja: (A) AOAC resmi metode 2007.01 (buffered); (B) NH4Cl78 M.Á. González-Curbelo et al. / J. Chromatogr. 1358 (2014) 75-84garam-out (unbuffered); (C) NH4O2CH (buffered); dan (D) NH4OAc(buffered). Setiap metode mengharuskan langkah-langkah berikut: (1) 15 gramsampel (5 g gandum butir) diletakkan ke dalam polypropylene 50 mLCentrifuge tabung, dan benteng dibuat untuk menghasilkan 10, 75 dan400 ng g untuk analytes pestisida dan 200 ng g untuk IS (tabung yang vortexed untuk 1 min untuk lebih mengintegrasikan paku kesampel); (2) 15 mL MeCN yang mengandung 1% (v/v) selesai yangditambahkan untuk versi AOAC 2007.01 dan NH4OAc, atau 15 mLdari MeCN yang mengandung asam format 5% (v/v) untuk versi NH4O2CH, atau 15 mL MeCN untuk versi NH4Cl; (3) tabung yangterguncang keras oleh tangan untuk 1 min (gandum, 15 mL air adalahjuga ditambahkan dan sampel yang terguncang untuk 1 h sesuai denganstudi sebelumnya [22] menggunakan shaker otomatis); (4) 6 g anh.MgSO4plus 1.5 g anh. NaOAc ditambahkan untuk AOAC 2007.01Versi, atau 7.5 g NH4Cl, NH4O2CH, atau NH4OAc untuk yang lainVersi masing-masing; (5) tabung yang keras terguncang lagiuntuk 1 menit tangan dan disentrifugasi di 3700 rcf untuk 1 min; (6) 1 mLdari MeCN ekstrak dipindahkan ke d-SPE tabung berisianh 150 mg. MgSO4, 50 mg PSA (150 mg untuk gandum), 50 mg C18, dan7.5 mg GCB, vortexed untuk 30 s, dan disentrifugasi di 3700 rcf untuk1 menit; (7) 0.5 mL ekstrak dipindahkan ke cawan autosampler tepat berlabel, dan 50 L (17 L gandum)solusi QC ditambahkan (untuk membuat semua sampel volume sama denganStandar Kalibrasi cocok matriks, 50 atau 17 L dari MeCNjuga ditambahkan yang diperlukan); (8) untuk LPGC-MS/MS, 200 L masing-masingekstrak telah dipindahkan ke dalam kedua autosampler botol berisidinonaktifkan sisipan kaca serta 20 L larutan protectants analyte; (9) untuk LC-MS/MS, 600 L larutan 0,1% formiclarutan asam ditambahkan ke L 300 dari ekstrak tersisa divial pertama. Untuk lemon, percepatan muncul setelah pengenceran denganair, dan semua ekstrak akhir disaring melalui 0.45 mPVDF Mini-UniPrep filter cawan dari Whatman (Florham Park, NJ,USA).Kalibrasi cocok matriks terlibat langkah yang sama seperti di atasmenggunakan ekstrak kosong, kecuali langkah d-SPE (6) adalah ditingkatkan8 ml dalam 15 mL tabung dan ada lonjakan atau adalah solusi yangditambahkan sampai langkah (7), ketika 50 L larutan standar kalibrasi (17 L untuk gandum butir) ditambahkan ke ekstrak dalambotol autosampler. Digunakan sebagai IS LPGC-MS/MS Fenthion-d6wasdan atrazine-d5for LC-MS/MS. Dalam kasus NH4O2CH akhirmetode buffered, tambahan validasi eksperimen dilakukan, dan hasil dibandingkan menggunakan langkah d-SPE (6)atau tidak.3. hasil dan diskusiSeperti yang ditunjukkan dalam tabel 1, total 48 ditargetkan kimia termasukIS, QC, isomer, adducts, dan degradasi produk dimonitor di LPGC-MS/MS dan LC-MS/MS dengan 15 tumpang tindih. Thealdicarb Na-adisi dipantau karena penasaran dalam studi, dantetrahydrophthalimide (THPI) dan phthalimide (PI) adalah produk degradasi captan dan folpet, masing-masing. Meskipunhanya orang tua analytes ditambahkan ke dalam campuran, THPI danPI bisa semi diukur dari kehadiran mereka di standar kalibrasi. Namun, captan dan folpet ketat diperlakukansebagai analytes pestisida, tidak THPI dan PI. Juga, spinosad referensi standar terdiri dari 85% spinosyn A dan spinosyn 15% D,dan meskipun keduanya telah dimonitor dan dilaporkan secara independen,mereka diperlakukan sebagai satu pestisida analyte (spinosad). Themetabolit sulfone aldicarb dan fenthion masing-masing disusundari standar terpisah, dan mereka semua diperlakukan sebagai unikanalytes. Isomer 4 dari Sipermetrin telah terintegrasi sebagai sebuah puncak tunggal. Oleh itu, terdapat analytes berbeda pestisida 43 dimetode.Pestisida yang hati-hati dipilih untuk mencakup berbagaidari perwakilan analytes kelas kimia yang berbeda biasanyadipantau di banyak labs di seluruh dunia. Dengan beberapa perbedaan kecil, ini telah digunakan dalam studi sebelumnya untuk menunjukkanmetode kinerja [6,15,18], dan telah menunjukkan bahwaKetika hasil yang dapat diterima untuk pestisida ini tercapai, makametode ini menghasilkan kualitas yang sama hasil untuk ratusan pestisida lain sifat yang sama [6,7,23]. Memang, jaminan kualitasPedoman merekomendasikan penggunaan pestisida perwakilan danmatriks seperti ini untuk kontrol kualitas paku selama rutinAnalisis [24].3.1. optimasi rasio garam asam amonium untuk penyanggaDalam studi sebelumnya oleh Nanita dan Padivitage [11], amoniumklorida, asetat, formate, karbonat dan sulfat dievaluasiuntuk memilih pengasinan terbaik keluar reagen untuk ekstraksi di QuEChERS sebelum FI-MS/MS. Hanya 14 pestisida dimonitor, Semuayang merupakan LC-setuju (tidak ada yang berada hanya GC-kompatibel), dan Penyanggaan tidak dilakukan. NH4Cl, NH4O2CH, danNH4OAc semua juga memberikan hasil yang dapat diterima dalam percobaan,dan NH4Cl pada akhirnya dipilih terutama karena pertimbangan praktis kecil dengan biaya yang sedikit lebih rendah. Namun, NH4Cl benar-benartelah mencair agak tinggi dan titik didih 338 dan 520◦C,masing-masing, dan sublimes untuk membentuk amonia dan HCl. PadaSebaliknya, NH4O2CH, dan NH4OAc memiliki titik leleh yang hanya110–117◦C and boiling points of 165–180◦C, and upon heating,they degrade to water plus formamide and acetamide, respectively, rather than direct generation of more corrosive products.Furthermore, buffering at pH 4–5 in QuEChERS had been previously demonstrated to yield higher recoveries for pH-dependentpesticides [7,15], and initial experiments were designed to determine the amount of HOAc and formic acid that should be used toachieve a suitable pH in combination with their respective ammonium salts.In this experiment, 15 g of apple and lemon samples were separately extracted using 15 mL of MeCN with increasing amounts ofHOAc or formic acid (1, 2, 3, 5, 7.5 or 10%, v/v) plus 7.5 g of NH4OAcor NH4O2CH, respectively (we used 1:1 sample:solvent ratio andexcess solids rather than saturated aqueous solutions employed byNanita and Padivitage [11]). After agitation and centrifugation, thetwo phases were separated and pH was measured in both layersincluding 4-fold dilution with deionized water for the MeCN phaseas done previously [15]. Fig. 1 shows the pH results from the experiment. As expected, the formate buffer (pKa= 3.75) provided lowerpH than the acetate buffer (pKa= 4.75), and good consistency forboth matrices. The addition of 5% formic acid yielded a pH valuearound 3 in the MeCN phase and